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TITLE:
ETHYL
TERTIARY
BUTYL
ETHER
ORAL
ABSORPTION
IN
MALE
AND
FEMALE
RATS
SPONSOR:
Section
211(
b)
Research
Group
American
Petroleum
Institute
1220
L
Street
NW
Washington,
DC
20005
TESTING
FACILITY:
Science
and
Engineering
RTI
International*
Post
Office
Box
12194
3040
Cornwallis
Road
Research
Triangle
Park,
NC
27709
RTI
PROJECT
NO.:
09408.007
RTI
STUDY
DIRECTOR:
Timothy
R.
Fennell
PROPOSED
EXPERIMENTAL
START
DATE:
April
2005
PROPOSED
EXPERIMENTAL
TERMINATION
DATE:
January
2006
SPONSOR
REFERENCE
NO:

AMENDMENTS:

No.
Date
Section
Pages
1
2
3
4
Thomas
E
Gray,
MS,
DABT
Sponsor's
Representative
Date
Timothy
R.
Fennell,
Ph.
D.
Study
Director
RTI
International
Date
RTI
International
is
a
trade
name
of
Research
Triangle
Institute.
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Table
of
Contents
Page
1.0
OBJECTIVES.............................................................................................................................
4
2.0
PERSONNEL..............................................................................................................................
4
3.0
STUDY
DESIGN.........................................................................................................................
4
4.0
JUSTIFICATIONS
......................................................................................................................
5
4.1
Animal
Species
...........................................................................................................................
5
4.2
Numbers
of
Animals....................................................................................................................
5
4.3
Routes
of
Administration
and
Dose
Levels
..................................................................................
5
5.0
REGULATORY
COMPLIANCE
..................................................................................................
5
6.0
TEST
SUBSTANCE
...................................................................................................................
5
7.0
ANIMALS....................................................................................................................................
6
7.1
Husbandry...................................................................................................................................
6
7.1.1
Identification...................................................................................................................
7
7.1.2
Quarantine......................................................................................................................
7
7.1.3
Feed
and
Water..............................................................................................................
7
7.1.4
Environmental
................................................................................................................
7
7.1.5
Acclimation
and
Housing
During
Studies
........................................................................
7
7.2
Randomization
and
Assignment
of
Animals
to
Treatment
Groups
...............................................
7
7.3
Body
Weights..............................................................................................................................
8
7.4
Found
Dead/
Moribund
Animals
...................................................................................................
8
7.5
Anesthesia
and
Euthanasia
.........................................................................................................
8
8.0
STUDY
PROCEDURES..............................................................................................................
8
8.1
Test
Chemical
Preparation
and
Analysis
.....................................................................................
8
8.2
Cannulation
of
Rats
for
Blood
Collection
.....................................................................................
9
8.3
Dosing.........................................................................................................................................
9
8.4
Ante
mortem
Observations
and
Functional
Assessments
............................................................
9
8.5
Collection
and
Storage
of
Biological
Samples
...........................................................................
10
8.5.1
Blood............................................................................................................................
10
8.6
Analysis
of
Blood
Samples.
.......................................................................................................
10
9.0
PHARMACOKINETIC
ANALYSIS
............................................................................................
10
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10.0
STATISTICAL
ANALYSIS........................................................................................................
11
11.0
DATA
FROM
"
EXTRA"
ANIMALS
...........................................................................................
11
12.0
RECORDS
AND
REPORT........................................................................................................
11
13.0
MAINTENANCE
OF
RECORDS
AND
RAW
DATA...................................................................
12
14.0
SAFETY
PRECAUTIONS.........................................................................................................
13
15.0
PROTOCOL
AMENDMENTS
AND
DEVIATIONS
....................................................................
13
16.0
REFERENCES
.........................................................................................................................
13
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1.0
OBJECTIVES
The
objectives
of
this
study
on
ethyl
tertiary
butyl
ether
(
ETBE)
are
to:

Develop
a
method
for
the
analysis
of
ETBE
in
blood
by
headspace
GC
Conduct
an
evaluation
of
oral
absorption
by
measuring
ETBE
in
blood
in
male
and
female
Fischer
344
rats
administered
ETBE
by
gavage
at
one
of
two
dose
levels,
10
and
100
mg/
kg.

2.0
PERSONNEL
 
Timothy
R.
Fennell,
Ph.
D.,
Study
Director
 
Susan
Sumner,
Ph.
D.,
Chemist
 
Norman
Gaudette,
B.
S.
 
Research
Chemist
 
Rodney
Snyder,
M.
S.
 
Research
Chemist
 
Yan
Hong,
M.
S
Research
Chemist
 
Jem
Scott­
Emuakpor,
DVM
­
Veterinarian
 
Melody
Gower
 
Biologist
Other
personnel
will
be
used
as
required.
A
full
list
of
study
participants
will
be
included
in
the
study
report.

3.0
STUDY
DESIGN
For
the
pharmacokinetic
analysis
of
ETBE,
a
GC
method
will
be
developed
for
the
quantitation
of
ETBE
in
blood.
To
provide
rat
blood
for
development
of
the
method,
up
to
thirty
male
rats
will
be
sacrificed
for
the
collection
of
control
blood.
The
rats
will
be
euthanized
under
CO2
as
needed
on
the
study,
and
exanguinated
by
cardiac
puncture.

For
the
analysis
of
blood
concentrations,
all
time­
point
blood
samples
from
ETBE
exposures
will
placed
in
glass
crimp
seal
vials
and
sealed.
Control
blood
samples
will
be
used
to
develop
a
GC
method
for
analyzing
the
concentration
of
ETBE
in
blood.
Once
the
method
is
verified,
blood
samples
will
be
used
to
prepare
a
standard
curve.
Aliquots
of
standards
will
be
frozen
at
approximately
­
20
o
C
or
below
to
provide
sets
of
standard
curve
solutions
for
the
definitive
pharmacokinetic
studies.
Details
of
the
method
will
be
recorded
in
the
raw
data.

ETBE
will
be
administered
in
water
at
a
dose
of
10
or
100
mg/
kg.
For
each
dose
level,
six
male
rats
and
six
female
rats
will
be
cannulated
with
jugular
vein
cannulas
up
to
4
days
prior
to
dosing
and
the
cannulas
will
be
kept
patent.
Two
additional
male
rats
and
two
additional
female
rats
will
be
cannulated
and
kept
on
hand
in
the
event
that
a
cannula
fails.
One
additional
male
rat
and
one
additional
female
rat
will
be
available
in
the
event
of
an
accidental
death
prior
to
cannulation.
ETBE
will
be
administered
by
gavage
in
water.
The
syringe,
needle
and
contents
will
be
weighed
prior
to
dosing,
and
after
dosing
the
weight
of
the
empty
syringe
and
needle
will
be
recorded.
The
weight
of
the
dose
administered
will
be
calculated.

The
only
biological
samples
collected
for
analysis
in
this
study
will
be
blood.
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It
is
anticipated
that
the
study
will
be
conducted
with
male
rats
administered
100
mg/
kg
ETBE
on
day
1,
female
rats
administered
100
mg/
kg
on
day
2.
Analysis
of
the
samples
from
this
part
of
the
study
will
be
conducted
prior
to
the
start
of
the
second
portion
of
the
study
approximately
1
week
later
in
which
male
rats
will
be
dosed
with
10
mg/
kg
ETBE
on
day
8
and
female
rats
will
be
dosed
with
10
mg/
kg
ETBE
on
day
9.
Male
and
female
rats
will
be
ordered
specifically
for
each
dose
group,
and
thus
will
not
be
randomly
assigned
to
a
treatment
group.

4.0
JUSTIFICATIONS
4.1
Animal
Species
The
present
studies
are
designed
to
evaluate
pharmacokinetics
of
ETBE
to
provide
information
that
will
be
used
for
safety
assessments
to
humans.
No
in
vitro
techniques
are
available
that
allow
for
adequate
determination
of
uptake,
distribution,
and
excretion
of
chemicals
by
mammals.
Fischer
344
rats
are
an
established
animal
species
and
strain
for
toxicological
testing,
and
pharmacokinetic
studies.

4.2
Numbers
of
Animals
The
numbers
of
animals
used
in
this
study
are
considered
acceptable
to
develop
the
analytical
procedures,
and
to
evaluate
the
appropriateness
of
the
exposure
generation
system
for
further
study.

4.3
Routes
of
Administration
and
Dose
Levels
The
route
of
administration
is
one
of
the
expected
exposure
routes
in
humans
(
through
ground
water).
Oral
administration
is
commonly
used
in
toxicity
or
safety
assessment
studies.
The
dose
vehicle,
water,
corresponds
to
a
potential
human
exposure
scenario.
The
doses
are
10
and
100
mg/
kg,

and
are
expected
to
be
without
significant
toxicity.
The
high
dose
was
selected
based
on
the
concentration
of
ETBE
that
can
be
reasonably
achieved
in
a
dose
volume
of
10
ml/
kg.
The
solubility
of
ETBE
in
water
is
1.2
%,
which
is
expected
to
enable
preparation
of
a
solution
of
10
mg/
ml
for
gavage
dosing.

5.0
REGULATORY
COMPLIANCE
This
study
will
be
carried
out
in
compliance
with
the
U.
S.
EPA
Good
Laboratory
Practice
Standards,
40
CFR
part
792.

6.0
TEST
SUBSTANCE
NAME:
Ethyl
tertiary
Butyl
Ether
(
ETBE;
tertiary
butyl
ethyl
ether,
2­
ethoxy­
2­
methylpropane)

CAS
No.:
637­
92­
3
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MOLECULAR
FORMULA:
C6H14O
MOLECULAR
WEIGHT:
102.17
STRUCTURE:

CH3
C
H3C
H3C
O
H2
C
CH3
SOURCE
OF
TEST
SUBSTANCE:
ETBE
will
be
purchased
from
Sigma­
Aldrich,
Milwaukee,
WI
(
Catalog
number
253898,
specified
purity
99%).
A
certificate
of
analysis
will
obtained
from
the
vendor.

LOT
NUMBER(
S):
To
be
listed
in
the
final
report.

IDENTITY
AND
PURITY:
The
identity
of
the
unlabeled
ETBE
will
be
confirmed
at
RTI
by
1
H
and
13
C
NMR,
and
by
mass
spectrometry.
The
purity
of
the
test
chemical
will
be
determined
by
GC­
MS.

STORAGE
CONDITIONS:
ETBE
will
be
stored
in
the
dark
at
room
temperature.

.

7.0
ANIMALS
1.
Species
and
Strains:
Fischer
344
rats
2.
Approximate
Age:
8­
9
weeks
old
at
time
of
dosing.
(
Male
rats
that
are
used
as
blood
donors
for
analytical
methods
development
may
be
significantly
older
than
the
8­
9
weeks
of
age,
and
this
will
not
affect
the
study).

3.
Approximate
Weight:
200
g
4.
Number/
Sex:

Up
to
48
male
rats
and
18
female
rats.

5.
Sources:
Taconic
(
Germantown,
NY)
will
be
the
primary
source
of
animals.
In
the
event
that
suitable
animals
cannot
be
provided
from
the
primary
source,
acceptable
alternate
sources
are
Charles
River
Laboratories,
Inc.
(
Portage,
MI),
and
Harlan
(
Indianapolis,
IN).

The
source(
s)
of
all
animals
will
be
included
in
the
Study
Report.

7.1
Husbandry
Research
Triangle
Institute
is
accredited
by
AAALAC
International.
Animal
procedures
detailed
in
this
protocol
are
in
accordance
with
the
Animal
Welfare
Act,
"
Guide
for
the
Care
and
Use
of
Laboratory
Animals"
(
NRC,
1996),
and
the
Office
of
Laboratory
Animal
Welfare
(
NIH).
All
animal
procedures
will
be
reviewed
by
RTI's
Institutional
Animal
Care
and
Use
Committee
(
IACUC)
before
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initiation
of
the
studies.
In
the
opinion
of
the
Sponsor
and
Study
Director,
the
study
does
not
unnecessarily
duplicate
any
previous
work.

7.1.1
Identification
Rats
will
be
identified
by
individual
metal
ear
tags.
All
individual
animal
data
will
be
referenced
to
either
ear
tag
number
or
to
treatment
group
and
animal
number
or
to
both.

7.1.2
Quarantine
Animals
will
be
quarantined
for
a
minimum
of
five
days
before
use
on
a
study.
Animals
will
be
examined
by
a
veterinarian
or
their
representative
prior
to
their
release
from
quarantine,
and
only
animals
determined
to
be
in
good
health
as
indicated
by
body
weight
gain
and
the
absence
of
clinical
signs
will
be
used.
During
the
quarantine
period
and
prior
to
initiation
of
the
experiments
detailed
in
Section
8.0,
rats
will
be
housed
(
maximum
of
3
per
cage)
in
polycarbonate
cages
with
stainless
steel
bar
lids
accommodating
a
water
bottle.
Cage
sizes
are
approximately
19"
x
10
1/
2"
x
8"
high
(
ca.
143
sq.
in.

floor
space).
Contact
bedding
will
be
Sani­
Chips
(
P.
J.
Murphy
Corporation,
Montville,
New
Jersey).

7.1.3
Feed
and
Water
Animals
will
be
provided
Certified
Purina
Rodent
Chow
(
5002)
ad
libitum.
Water
will
be
provided
ad
libitum.
The
source
of
the
water
is
the
City
of
Durham,
NC.
The
analysis
of
water
and
analysis
of
the
rodent
chow
for
chemical
composition
and
possible
chemical
contamination
will
be
provided
by
the
suppliers
and
maintained
in
the
Study
Records.
It
is
anticipated
that
contaminant
levels
will
be
below
those
permitted
in
the
certified
feed
and
will
not
affect
the
design,
conduct,
or
conclusions
of
this
study.

7.1.4
Environmental
Air
circulation
will
be
100%
fresh
air.
Room
temperature
will
be
maintained
at
64 
79
°
F
and
relative
humidity
at
30 
70%
and
monitored
at
least
once
a
day.
Light/
darkness
will
be
cycled
at
12­
h
intervals.
Any
deviations
from
these
conditions
shall
be
included
in
the
study
records.
Environmental
parameters
will
be
recorded
automatically
using
a
computerized
HVAC
Monitoring
and
Control
System.

7.1.5
Acclimation
and
Housing
During
Studies
Animals
will
be
acclimated
for
a
minimum
of
5
days
prior
to
use
on
studies.
Animals
will
be
housed
in
polycarbonate
cages
with
stainless
steel
lids.
Following
administration
of
ETBE,
the
cages
will
be
placed
in
a
hood
for
the
duration
of
the
post
dosing
period.

7.2
Randomization
and
Assignment
of
Animals
to
Treatment
Groups
Animals
designated
as
blood
donors
for
methods
development
will
not
be
randomized.
Animals
designated
for
dosing
with
ETBE
will
be
specifically
ordered
to
be
at
the
appropriate
age
and
body
weight
at
the
time
of
dosing.
To
accommodate
the
dosing
schedule,
separate
groups
of
9
male
and
female
rats
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will
be
purchased
for
each
dose
group.
For
each
dose
group,
a
total
of
9
rats
will
be
available
for
cannulation
prior
to
dosing.
After
cannulation,
and
before
dosing,
animals
within
each
treatment
group
will
also
be
assigned
in
numerical
order
to
sequential
values
listed
in
Table
VII
"
Random
Sampling
Numbers"
which
appears
in
the
Appendix
of
The
Statistical
Analysis
of
Experimental
Data,
by
John
Mandel,
Dover
Publications,
Inc.,
1964.
Assignment
of
an
animal
number
from
1­
9
will
be
conducted
by
ranking
the
random
numbers
in
ascending
order.
Animals
assigned
numbers
1 
4
in
the
treatment
groups
will
be
designated
as
"
core"
members
of
the
group;
animals
assigned
higher
numbers
will
be
designated
as
"
extra"
animals
(
see
Section
11.0
for
an
explanation
of
the
use
of
the
"
extra"
animals).

7.3
Body
Weights
Individual
body
weights
will
be
measured
during
the
quarantine
period,
the
day
of
exposure,
and
at
sacrifice.

7.4
Found
Dead/
Moribund
Animals
The
Study
Director
or
the
veterinarian
will
authorize
euthanasia
of
animals
with
life­
threatening
clinical
signs
that
indicate
that
they
are
unlikely
to
survive
until
the
next
scheduled
observation.
The
time
of
death
will
be
estimated
as
precisely
as
possible
and
recorded.

7.5
Anesthesia
and
Euthanasia
Anesthesia
will
be
used
to
avoid
undue
pain
or
distress.
Animals
will
be
anesthetized
with
CO2
exposure
just
prior
to
study
termination.
Rats
will
be
euthanized
by
exsanguination.

8.0
STUDY
PROCEDURES
8.1
Test
Chemical
Preparation
and
Analysis
ETBE
will
be
prepared
for
gavage
dosing
as
a
solution
in
water.
For
dosing
of
animals,
the
dose
solution
will
be
prepared
within
24
hours
of
dosing,
and
maintained
at
room
temperature.
The
nominal
concentration
will
be
calculated
from
the
weight
of
ETBE
and
the
weight
of
water
added
to
the
dose
preparation.
Dose
solutions
will
be
prepared
at
two
concentrations,
the
first
at
approximately
10
mg/
ml
and
the
second
at
1
mg/
ml,
to
ensure
that
the
volume
of
dose
administered
per
kg
body
weight
is
similar
in
the
high
and
the
low
dose
groups.
Prior
to
dosing,
a
method
will
be
prepared
for
the
quantitative
analysis
of
ETBE
in
water,
by
dissolving
a
weighed
aliquot
of
the
ETBE/
water
solution
in
a
suitable
solvent
such
as
methanol.
Aliquots
of
this
solution
will
be
analyzed
directly
by
GC,
using
a
standard
curve
for
the
quantitation
of
ETBE.
The
stability
of
ETBE
in
water
will
be
determined
by
analysis
of
aliquots
of
a
mock
dose
solution
of
ETBE
kept
at
room
temperature
for
three
days.
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When
appropriate
methods
have
been
developed
for
the
detection
of
ETBE
in
blood,
and
for
the
quantitative
analysis
of
ETBE
in
dosing
solutions,
and
the
stability
of
ETBE
in
blood
and
dose
solution
have
been
established,
the
animal
study
will
be
conducted.

8.2
Cannulation
of
Rats
for
Blood
Collection
Rats
in
the
ETBE
treatment
groups
will
be
prepared
with
an
indwelling
jugular
cannula
to
facilitate
painless,
nontraumatic
collection
of
serial
blood
samples.
The
surgical
technique
will
be
similar
to
that
reported
by
McKenna
and
Bieri
(
1984).
Animals
will
be
anesthetized
with
ketamine/
xylazine
(
7:
1,

60­
80
mg/
kg
or
to
effect).
A
small
incision
at
the
level
of
the
clavicle
approximately
0.5
cm
from
the
midline
will
afford
access
to
the
right,
external
jugular
vein.
The
vessel
will
be
blunt
dissected
and
the
cannula
(
prefilled
with
sterile
saline
containing
20
IU/
mL
of
sodium
heparin)
will
be
inserted
to
a
depth
of
approximately
28
mm.
Placement
in
the
atrium
will
be
confirmed
by
patency.
The
cannula
will
be
secured
in
the
vessel
at
the
insertion
point
by
sutures
to
the
surrounding
musculature.
The
cannula
will
then
be
exteriorized
by
passing
the
distal
end
subcutaneously
around
the
neck
to
exit
the
dorsal
surface
via
a
small
incision
between
the
scapulae.
The
ventral
incision
will
be
closed
with
sutures.
The
distal
end
of
the
cannula
will
be
sutured
to
the
base
of
the
neck
between
the
scapulae.
Animals
will
be
allowed
to
recover
a
minimum
of
two
days
prior
to
dosing.
Cannula
patency
will
be
maintained
with
daily
flushings
of
heparinized
saline.
At
least
six
rats
for
each
dose
group
will
be
prepared
with
cannulas.

8.3
Dosing
Each
rat
will
be
weighed
prior
to
dosing
to
determine
the
amount
of
dose
to
be
administered.

Gavage
doses
will
be
administered
using
a
syringe
fitted
with
a
blunt
tipped
gavage
dosing
needle.
The
dose
administered
to
each
animal
will
be
determined
from
the
weight
of
the
full
syringe
minus
that
of
the
empty
syringe.
The
dose
time
will
be
recorded.
Dosing
of
animals
will
be
spaced
apart
to
allow
blood
collection
at
the
appropriate
times
(
see
section
8.4.1
below).
The
groups
will
be
designated
according
to
the
dose
administered
and
sex
of
the
rats:
Group
A
male
100
mg/
kg,
Group
B
female
100
mg/
kg,
Group
C
male
10
mg/
kg,
Group
D
female
10
mg/
kg.
At
least
four
cannulated
rats
per
group
will
be
dosed
with
ETBE.

8.4
Ante
mortem
Observations
and
Functional
Assessments
Animals
will
be
observed
twice
per
day
for
mortality,
morbidity,
signs
of
toxicity,
and
for
any
acute
distress
that
might
be
related
to
the
test
procedure
or
test
substances.
Animals
exhibiting
adverse
reactions
will
be
closely
monitored.
All
signs
of
poor
health
or
abnormal
behavior
will
be
recorded.
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8.5
Collection
and
Storage
of
Biological
Samples
8.5.1
Blood
Blood
samples
will
be
collected
prior
to
dosing
from
each
rat
(
time
=
0),
and
from
4
rats
of
each
sex
and
at
each
dose
at
15
and
30
min,
and
at
1,
2,
4,
8,
and
24
hr
after
dosing.

Blood
samples
will
be
drawn
from
the
jugular
vein
cannula
into
a
heparinized
1
ml
syringe,
and
the
sample
will
be
immediately
placed
in
a
preweighed
headspace
vial.
The
amount
of
blood
will
be
approximately
100
µ
l.
The
vial
will
be
immediately
crimped
and
weighed,
and
the
weight
of
the
blood
aliquot
will
be
determined.
After
the
last
sample
has
been
collected
from
each
animal
at
24
hr,
the
rats
will
be
euthanized
by
exposure
to
CO2.
Should
cannulas
fail
between
the
8
and
24
hr
blood
samples,

blood
will
be
collected
by
cardiac
puncture
under
CO2
anesthesia
at
sacrifice
at
24
hr.
The
blood
samples
will
be
maintained
at
room
temperature
until
analyzed.
It
is
anticipated
that
blood
samples
will
be
analyzed
by
GC
within
24
hours
of
collection.

Blood
samples
from
unexposed
rats
will
be
used
for
methods
development.
Blood
samples
will
be
collected
by
cardiac
puncture,
under
CO2
anesthesia,
and
the
rats
will
be
euthanized.

8.6
Analysis
of
Blood
Samples.

A
GC
method
will
be
developed
for
quantitating
ETBE
using
blood
from
control
male
Fischer
344
rats.
The
methods
will
involve
the
development
of
a
standard
curve
for
analysis
of
ETBE
in
the
headspace
of
air
tight
vials
containing
a
known
volume
of
control
blood
and
a
known
concentration
of
ETBE.
The
stability
of
ETBE
will
be
evaluated
under
storage
conditions
that
may
be
used
in
this
study
will
be
evaluated
during
this
method
development.
This
will
include
analysis
of
ETBE
immediately
spiking
in
control
blood,
after
storage
at
room
temperature
(
8
hr)
or
at
approximately
4
o
C
(
24
hr).
The
concentration
of
ETBE
in
the
headspace
of
vials
containing
a
known
volume
of
blood
from
rats
administered
ETBE
by
gavage
will
be
determined
by
comparison
to
the
standard
curve.

9.0
PHARMACOKINETIC
ANALYSIS
Individual
and
mean
blood
concentration­
time
data
for
each
dose
and
sex
will
be
analyzed,
as
appropriate,
by
noncompartmental
(
model­
independent)
using
the
least­
squares
fitting
program
WinNonlin
(
Statistical
Consulting
Inc.,
Cary,
North
Carolina).
The
following
pharmacokinetic
parameters
will
be
determined
as
appropriate:
terminal
elimination
rate
constant,
terminal
elimination
half­
life
(
T1/
2),

area
under
the
blood
concentration­
time
curve
extrapolated
from
time
zero
to
infinity
(
AUC),
maximum
concentration
achieved
(
Cmax),
and
time
to
maximum
concentration
(
Tmax).
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10.0
STATISTICAL
ANALYSIS
All
blood
concentration
data
will
be
reported
in
tables
as
the
mean
±
standard
deviation
(
SD).

Pharmacokinetic
data
will
be
presented
as
mean
±
SD.
The
methods
to
be
used
for
statistical
analysis
will
be
added
by
amendment.

11.0
DATA
FROM
"
EXTRA"
ANIMALS
To
better
ensure
that
complete
data
from
the
required
number
of
animals
in
each
treatment
group
will
be
obtained,
one
or
more
additional
animals,
predesignated
as
"
extras",
will
start
the
study
as
part
of
each
treatment
group.
If
a
"
core"
study
animal
fails
to
complete
the
study
due
to
loss
of
samples,

misdosing,
morbidity,
or
other
accidents,
it
will
be
replaced
with
a
designated
"
extra"
animal
and
data
will
cease
to
be
obtained
from
the
"
core"
animal.
The
"
extra"
animal
will
then
become
part
of
the
"
core"

group
and
the
original
animal
will
be
removed
from
the
"
core"
group.
All
such
substitutions
will
be
documented
as
to
reason
and
approved
in
writing
by
the
Study
Director.
Except
for
animals
that
become
part
of
a
"
core"
group,
samples
from
"
extra"
animals
will
not
normally
be
analyzed.
However,
all
data
obtained
in
the
study
will
be
reported.
Data
used
to
construct
group
means
and
to
obtain
pharmacokinetic
parameters
will
consist
of
the
data
obtained
from
the
"
core"
study
animals
(
not
the
"
extras")
except
in
cases
where
an
"
extra"
has
taken
the
place
of
a
"
core"
animal.
In
that
case,
the
data
from
the
"
extra"
animal
will
be
used
instead,
and
data
from
the
"
core"
animal
that
was
eliminated
from
the
study
will
not
be
used
in
these
calculations.

12.0
RECORDS
AND
REPORT
The
following
will
be
maintained
in
the
record:

a.
Protocol
and
any
amendments
b.
Animal
receipt
records
c.
Quarantine
records
d.
Temperature
and
humidity
records
for
the
treatment
rooms
e.
Animal
research
facility
room
logs
f.
Feed
and
water
analysis
for
contaminants
g.
Randomization
records
h.
Test
chemical
receipt,
storage
and
use
records
i.
Balance
calibration
log
references
j.
Correspondences
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k.
All
other
raw
data
and
documentation.

Results
of
the
studies
will
be
described
in
an
audited
draft
report,
which
will
be
submitted
to
the
Sponsor
for
approval.
This
report
will
include
but
not
be
limited
to:

a.
Name
and
address
of
the
facility
performing
the
study,
dates
of
study
initiation
and
completion,
and
RTI
study
number.

b.
A
copy
of
the
signed,
dated
and
approved
protocol
and
all
deviations
and
authorized
amendments
to
the
original
protocol.

c.
A
detailed
description
of
all
methods
used,
including
the
randomization
method.

d.
The
lot
number(
s)
of
the
test
substances
and
details
of
the
formulation
of
doses.

e.
Animal
information
to
include:
supply
source,
species,
strain
or
substrain,
sex,
individual
animal
weights
(
at
randomization
through
sacrifice),
approximate
age
at
initiation
of
dosing,
and
procedure
used
for
individual
animal
identification
and
assignment
to
the
treatment
group.

f.
Tabulated
individual
results
for
blood
concentration.

g.
Tabulated
mean
results
for
blood
concentration.

h.
Pharmacokinetic
and
statistical
analysis
of
the
data.

Upon
acceptance
of
the
draft
report
by
the
Sponsor,
a
final
report
will
be
issued.
Two
bound
and
one
unbound
copies
of
the
audited
draft
report
and
two
bound
and
one
unbound
copies
of
the
final
report
will
be
shipped
to:

Matthew
Todd
Regulatory
Analysis
and
Scientific
Affairs
American
Petroleum
Institute
1220
L
Street
NW
Washington,
DC
20005
p:
(
202)
682­
8319
f:
(
202)
682­
8031
13.0
MAINTENANCE
OF
RECORDS
AND
RAW
DATA
Records
will
be
maintained
in
the
laboratories
of
the
study
personnel
while
the
studies
are
being
conducted.
Raw
data
generated
while
conducting
the
study
and
any
transformations,
calculations
or
operations
performed
on
the
data
will
be
recorded
in
the
study
file.
All
raw
data,
documentation,

records,
protocols,
amendments,
and
the
final
report
will
be
stored
in
the
RTI
International
Archives,

under
the
control
of
the
RTI
Quality
Assurance
Unit
in
accordance
with
FDA,
EPA
FIFRA
GLP
regulations
and
OECD
Principles
of
GLP.
The
applicable
record
retention
requirements
for
this
study
are
those
of
EPA
TSCA
GLPs,
40
CFR
part
792,
subpart
J.
Documentation
and
raw
data
will
be
maintained
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in
the
Archives
for
a
period
of
ten
years.
The
test
substance
ETBE
is
an
ether
that
can
generate
explosive
peroxides
on
storage.
Therefore,
an
archived
sample
of
test
chemical
will
not
be
retained.

The
samples
of
blood
collected
will
not
afford
analysis
beyond
the
duration
of
the
study.
Wet
specimens
of
blood
will
be
disposed
of
after
quality
assurance
verification
(
after
the
QAU
assures
that
discarding
the
samples
does
not
negatively
impact
the
integrity
of
the
study).

14.0
SAFETY
PRECAUTIONS
ETBE
is
a
flammable
liquid,
with
the
potential
to
form
explosive
peroxides.
The
container
should
be
tightly
closed
when
not
in
use.
Storage
under
a
nitrogen
atmosphere
is
recommended
to
avoid
the
generation
of
peroxides.
Use
personal
protective
equipment,
including
safety
glasses,
lab
coat,
and
chemical
resistant
gloves.

15.0
PROTOCOL
AMENDMENTS
AND
DEVIATIONS
This
protocol
may
be
amended
by
the
Study
Director
with
agreement
of
the
Sponsor
as
the
study
progresses.
Normally,
a
formal
amendment
will
be
prepared
and
signed
by
the
Study
Director
and
the
Sponsor's
Representative
prior
to
the
change.
If
instances
arise
where
a
change
is
urgent,
the
change
may
become
effective
upon
approval
by
the
Study
Director.
A
notification
of
the
urgent
change
will
be
sent
to
the
Sponsor's
Representative
(
email,
facsimile,
or
telephone)
as
soon
as
feasible
(
no
more
than
24
h
after
the
Study
Director's
approval).
Subsequently,
a
formal
protocol
amendment
will
be
prepared
for
approval
by
the
Study
Director
and
the
Sponsor's
Representative.

Any
deviations
from
the
protocol
that
occur
in
the
course
of
the
conduct
of
the
study
will
be
documented.
The
cause
for
the
deviation
and
its
effect
on
the
outcome
of
the
study
will
be
explained
and
the
Study
Director
will
sign
the
document.

16.0
REFERENCES
Harms
PG
and
Ojeda
SR
(
1974)
A
rapid
and
simple
procedure
for
chronic
cannulation
of
the
rat
jugular
vein.
J.
Appl.
Physiol.
36,
391­
392.

Mandel
J
(
1964)
The
Statistical
Analysis
of
Experimental
Data.
Dover
Publications,
Inc.

McKenna
MC
and
Bieri
JG
(
1984)
Multilayer
cannula
for
long­
term
infusion
of
unrestrained
rats.
Lab.

Animal
Sci.
34,
308­
310.

National
Research
Council
(
1996).
Guide
for
the
Care
and
Use
of
Laboratory
Animals.
National
Academy
Press:
Washington,
DC.
