Document ID: EPA-HQ-OPP-2003-0302-0024
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-12-23T05:00Z

SAP
Minutes
No.
2003­
03
October
28
and
29,
2003
FIFRA
Scientific
Advisory
Panel
Meeting,
Held
at
the
Holiday
Inn
Hotel,
Arlington,
Virginia
A
Set
of
Scientific
Issues
Being
Considered
by
the
Environmental
Protection
Agency
Regarding:

Ensuring
Data
Quality
for
In
Vitro
Tests
Used
as
Alternatives
to
Animal
Studies
for
Regulatory
Purposes:
A
Consultation
Myrta
R.
Christian,
M.
S.
Steven
G.
Heeringa,
Ph.
D.
Designated
Federal
Official
FIFRA
SAP,
Session
Chair
FIFRA
Scientific
Advisory
Panel
FIFRA
Scientific
Advisory
Panel
Date:
December
22,
2003
Date:
December
22,
2003
2
of
22
NOTICE
These
meeting
minutes
have
been
written
as
part
of
the
activities
of
the
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(
FIFRA),
Scientific
Advisory
Panel
(
SAP).
These
meeting
minutes
represent
the
views
and
recommendations
of
the
FIFRA
SAP,
not
the
United
States
Environmental
Protection
Agency
(
Agency).
The
content
of
these
meeting
minutes
do
not
represent
information
approved
or
disseminated
by
the
Agency.
These
meeting
minutes
have
not
been
reviewed
for
approval
by
the
Agency
and,
hence,
the
contents
of
this
report
do
not
necessarily
represent
the
views
and
policies
of
the
Agency,
nor
of
other
agencies
in
the
Executive
Branch
of
the
Federal
government,
nor
does
mention
of
trade
names
or
commercial
products
constitute
a
recommendation
for
use.

The
FIFRA
SAP
is
a
Federal
advisory
committee
operating
in
accordance
with
the
Federal
Advisory
Committee
Act
and
was
established
under
the
provisions
of
FIFRA,
as
amended
by
the
Food
Quality
Protection
Act
FQPA
of
1996.
The
FIFRA
SAP
provides
advice,
information,
and
recommendations
to
the
Agency
Administrator
on
pesticides
and
pesticide­
related
issues
regarding
the
impact
of
regulatory
actions
on
health
and
the
environment.
The
Panel
serves
as
the
primary
scientific
peer
review
mechanism
of
the
EPA,
Office
of
Pesticide
Programs
(
OPP)
and
is
structured
to
provide
balanced
expert
assessment
of
pesticide
and
pesticide­
related
matters
facing
the
Agency.
Food
Quality
Protection
Act
Science
Review
Board
members
serve
the
FIFRA
SAP
on
an
ad
hoc
basis
to
assist
in
reviews
conducted
by
the
FIFRA
SAP.
Further
information
about
FIFRA
SAP
reports
and
activities
can
be
obtained
from
its
website
at
http://
www.
epa.
gov/
scipoly/
sap/
or
the
OPP
Docket
at
(
703)
305­
5805.
Interested
persons
are
invited
to
contact
Larry
Dorsey,
SAP
Executive
Secretary,
via
e­
mail
at
dorsey.
larry@.
epa.
gov.

In
preparing
these
meeting
minutes,
the
Panel
carefully
considered
all
information
provided
and
presented
by
the
Agency
presenters.
This
document
addresses
the
information
provided
and
presented
within
the
structure
of
the
charge
by
the
Agency.
3
of
22
CONTENTS
PARTICIPANTS...........................................................................................................
4
INTRODUCTION.........................................................................................................
6
CHARGE
......................................................................................................................
7
PANEL
DELIBERATIONS
AND
RESPONSE
TO
CHARGE
..................................
9
REFERENCES............................................................................................................
20
4
of
22
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
Scientific
Advisory
Panel
Meeting
October
28
and
29,
2003
Ensuring
Data
Quality
for
In
Vitro
Tests
Used
as
Alternatives
to
Animal
Studies
for
Regulatory
Purposes:
A
Consultation
PARTICIPANTS
FIFRA
SAP,
Session
Chair
Steven
G.
Heeringa,
Ph.
D.,
Research
Scientist
&
Director
for
Statistical
Design,
University
of
Michigan,
Institute
for
Social
Research,
Ann
Arbor,
MI
Designated
Federal
Official
Mrs.
Myrta
R.
Christian,
FIFRA
Scientific
Advisory
Panel
Staff,
Office
of
Science
Coordination
and
Policy,
EPA
FIFRA
Scientific
Advisory
Panel
Members
Stephen
M.
Roberts,
Ph.
D.
(
FIFRA
SAP
Chair),
Professor
&
Program
Director,
University
of
Florida,
Center
for
Environmental
&
Human
Toxicology,
Gainesville,
FL
Stuart
Handwerger,
M.
D.,
Director,
Division
of
Endocrinology,
Cincinnati
Children's
Hospital
Medical
Center,
University
of
Cincinnati,
Cincinnati,
OH
Gary
E.
Isom,
Ph.
D.,
Professor
of
Toxicology,
School
of
Pharmacy
and
Pharmacological
Sciences,
Purdue
University,
West
Lafayette,
IN
Mary
Anna
Thrall,
D.
V.
M.,
Professor,
Department
of
Pathology,
College
of
Veterinary
&
Biomedical
Sciences,
Colorado
State
University,
Fort
Collins,
CO
FQPA
Science
Review
Board
Members
Joseph
P.
Bressler,
Ph.
D.,
Department
of
Neurobiology,
The
Kennedy
Krieger
Research
Institute,
Baltimore,
MD
George
L.
DeGeorge,
Ph.
D.,
Chief
Scientific
Officer,
MB
Research
Laboratories,
Spinnerstown,
PA
James
Freeman,
Ph.
D.,
ExxonMobil
Biomedical
Sciences,
Inc.,
Annandale,
New
Jersey
Michael
Luster,
Ph.
D.,
Chief,
Toxicology
and
Molecular
Biology
Branch,
National
Institute
for
Occupational
Safety
and
Health,
Morgantown,
WV
5
of
22
James
F.
McCormack,
Ph.
D.,
Director
of
Nonclinical
Laboratory
Compliance,
Food
&
Drug
Administration,
Rockville,
MD
Kenneth
Portier,
Ph.
D.,
Associate
Professor
of
Statistics,
Institute
of
Food
and
Agricultural
Sciences,
University
of
Florida,
Gainesville,
FL
Gregory
A.
Reed,
Ph.
D.,
Associate
Professor,
Department
of
Pharmacology,
Toxicology,
and
Therapeutics,
University
of
Kansas
Medical
Center,
Kansas
City,
KS
6
of
22
INTRODUCTION
The
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(
FIFRA),
Scientific
Advisory
Panel
(
SAP)
has
completed
its
review
of
the
set
of
scientific
issues
being
considered
by
the
Agency
pertaining
to
the
processes
for
regulatory
acceptance
of
and
ensuring
the
quality
of
data
from
in
vitro
tests
used
as
alternatives
to
animal
studies
for
regulatory
purposes.
Advance
notice
of
the
meeting
was
published
in
the
Federal
Register
on
September
22,
2003.
The
review
was
conducted
in
an
open
Panel
meeting
held
in
Arlington,
Virginia,
on
October
28
and
29,
2003.
Dr.
Steven
G.
Heeringa
chaired
the
meeting.
Mrs.
Myrta
R.
Christian
served
as
the
Designated
Federal
Official.

The
FIFRA
SAP
was
asked
to
review
issues
concerned
with
processes
for
regulatory
acceptance
of
and
ensuring
the
quality
of
data
from
in
vitro
tests
used
as
alternatives
to
animal
studies
for
regulatory
purposes,
including
performance
standards,
essential
test
method
components,
and
quality
control
of
test
methods,
in
the
context
of
three
new
in
vitro
assays
for
dermal
corrosivity
which
will
be
incorporated
into
its
OPTS
870.2500
test
guideline
for
Acute
Dermal
Irritation.

In
preparing
these
meeting
minutes,
the
Panel
carefully
considered
all
information
provided
and
presented
by
the
Agency
presenters,
as
well
as
information
presented
by
public
commenters.
These
meeting
minutes
address
the
information
provided
and
presented
at
the
meeting,
especially
the
response
to
the
charge
by
the
Agency.
7
of
22
CHARGE
Performance
Standards
The
Agency
plans
to
adopt
the
Performance
Standards
developed
by
the
Interagency
Coordinating
Committee
on
the
Validation
of
Alternative
Methods
(
ICCVAM)
as
a
means
of
communicating
the
basis
by
which
each
of
three
validated
in
vitro
test
methods,
Corrositex
®
,
EPISKIN
 
/
EpiDerm
 
,
and
Transcutaneous
Electrical
Resistance
(
TER),
are
deemed
acceptable
for
providing
dermal
corrosivity
data.
Performance
Standards
consist
of
descriptions
of
(
1)
essential
test
method
components,
which
are
the
essential
structural,
functional,
and
procedural
elements
of
a
validated
test
method
that
should
be
included
in
the
protocol
of
a
proposed
mechanistically
and
functionally
similar
test
method;
(
2)
a
minimum
list
of
Reference
Chemicals,
which
is
used
to
assess
the
accuracy
and
reliability
of
the
similar
test
method;
and
(
3)
comparable
accuracy
and
reliability
values
that
should
be
achieved
by
the
proposed
test
method
when
evaluated
using
the
minimum
set
of
Reference
Chemicals.

Question
1
Please
comment
on
the
provisions
in
the
Performance
Standards
for
each
of
the
three
methods
to
demonstrate
mechanistic
similarity
of
"
me­
too"
methods.
Do
the
essential
test
method
components
for
each
method
adequately
describe
the
unique
characteristics
of
the
method
necessary
to
determine
whether
a
test
is
mechanistically
and
functionally
similar?

Question
2
In
its
evaluation
of
any
mechanistically
similar
test
system,
the
Agency
plans
to
use
the
generic
criteria
used
by
ICCVAM
for
selecting
subsets
of
the
Reference
Chemicals
for
all
three
ICCVAM
Performance
Standards
documents.
The
criteria
specify
that
chemicals
should
be
selected
in
such
a
way
that
the
subset:
includes
representatives
of
applicable
chemical
classes,
measures
a
range
of
corrosive
strengths,
includes
well­
defined
chemicals
that
are
currently
available
commercially,
and
has
unequivocal
animal
or
other
in
vivo
evidence.
Please
comment
on
the
strengths
or
weaknesses
of
this
approach
and
identify
and
discuss
any
modifications
to
the
criteria
that
should
be
considered.

Question
3
The
ICCVAM
approach
for
demonstrating
functional
similarity
of
"
me­
too"
test
methods
to
validated
methods
includes
the
use
of
well­
characterized
Reference
Chemicals
and
specifies
the
accuracy
and
reliability
that
should
be
achieved
by
"
me­
too"
test
systems
when
tested
in
intra­
and
inter­
laboratory
studies.
Please
comment
on
whether
"
me­
too"
test
systems
should
be
demonstrated
to
be
effective
for
evaluating
the
testing
endpoint
for
all
of
the
chemicals
in
the
Performance
Standard.
Please
comment
on
the
value
of
including
chemicals
with
range
of
potencies
in
the
Performance
Standard.
Under
what
circumstances
might
testing
of
"
me­
too"
systems
within
one
laboratory
ever
be
sufficient
to
demonstrate
functional
equivalence?
8
of
22
Quality
Control
The
Agency
is
proposing
quality
control
measures
that
should
be
considered
when
evaluating
the
reliability
of
test
kits
for
regulatory
purposes.
Please
address
the
following
specific
issues.

Question
4
Subsets
of
the
Reference
Chemicals
used
in
test
method
validation
may
be
used
as
training
or
calibration
sets
by
testing
laboratories
using
in
vitro
systems.
Please
discuss
the
utility
of
and
necessity
for
training
or
calibration
sets
in
assuring
data
quality.
Please
comment
on
the
chemicals
selected
by
ICCVAM
for
use
as
a
calibration
set
for
TER
for
this
purpose.
Please
comment
on
the
ranges
of
chemical
classes
and
potencies
of
these
chemicals.
How
might
other
chemicals
be
selected
for
possible
use
in
the
calibration
sets?
Please
comment
on
the
value
of
identifying
chemicals
that
might
be
used
by
laboratories
as
training
sets
to
demonstrate
proficiency
in
performing
the
test.

Question
5
Anticipating
the
use
of
systems
using
tissue
constructs,
ex
vivo
systems,
microarrays
or
genetically
modified
cells,
please
discuss
aspects
of
the
quality
control
criteria
that
are
necessary
for
assuring
the
integrity
of
such
systems
over
time
and
from
lot­
to­
lot.
Please
comment
on
whether
and
how
the
type
of
system
­
tissue
constructs,
ex
vivo
systems,
or
genetically
modified
cells
or
animals
­
should
affect
the
criteria
for
quality
control
for
assuring
the
integrity
of
such
systems,
both
over
time
and
from
lot­
to­
lot.

Question
6
Please
comment
on
the
advantages
and
disadvantages
of
including
concurrent
positive
and
negative
controls
with
in
vitro
assays
when
used
as
alternatives
to
animal
testing.
What
are
the
important
characteristics
of
positive
and
negative
controls
for
in
vitro
studies?
What
aspects
of
positive
control
characteristics
allow
them
to
be
used
as
part
of
the
quality
control
process?
When
might
confirmation
that
positive
controls
are
performing
within
expected
or
historical
limits
be
sufficient
to
demonstrate
that
the
Proprietary
Test
Method
or
non­
proprietary
assay
system
is
functioning
properly?
When
might
additional
quality
control
measures
be
needed?

Question
7
Does
the
Panel
agree
that
the
benchmark
controls
serve
a
useful
purpose
to
demonstrate
the
level
of
response
that
can
be
expected
for
each
chemical
class
for
each
lot
of
Proprietary
Test
Method
assays?
Can
the
Panel
suggest
criteria
for
choice
of
appropriate
benchmark
controls?
9
of
22
PANEL
DELIBERATIONS
AND
RESPONSE
TO
CHARGE
The
specific
issues
addressed
by
the
Panel
are
keyed
to
the
Agency's
background
documents,
and
the
Agency's
charge
questions.

Response
to
Charge
I.
Performance
Standards
The
Agency
plans
to
adopt
the
Performance
Standards
developed
by
the
Interagency
Coordinating
Committee
on
the
Validation
of
Alternative
Methods
(
ICCVAM)
as
a
means
of
communicating
the
basis
by
which
each
of
three
validated
in
vitro
test
methods,
Corrositex
®
,
EPISKIN
 
/
EpiDerm
 
,
and
Transcutaneous
Electrical
Resistance
(
TER),
are
deemed
acceptable
for
providing
dermal
corrosivity
data.
Performance
Standards
consist
of
descriptions
of
(
1)
essential
test
method
components,
which
are
the
essential
structural,
functional,
and
procedural
elements
of
a
validated
test
method
that
should
be
included
in
the
protocol
of
a
proposed
mechanistically
and
functionally
similar
test
method;
(
2)
a
minimum
list
of
Reference
Chemicals,
which
is
used
to
assess
the
accuracy
and
reliability
of
the
similar
test
method;
and
(
3)
comparable
accuracy
and
reliability
values
that
should
be
achieved
by
the
proposed
test
method
when
evaluated
using
the
minimum
set
of
Reference
Chemicals.

Question
1
 
Please
comment
on
the
provisions
in
the
Performance
Standards
for
each
of
the
three
methods
to
demonstrate
mechanistic
similarity
of
"
me­
too"
methods.
Do
the
essential
test
method
components
for
each
method
adequately
describe
the
unique
characteristics
of
the
method
necessary
to
determine
whether
a
test
is
mechanistically
and
functionally
similar?

Panel's
comments:

The
Panel
endorsed
the
Performance
Standards
(
PS)
approach
to
identify
and
validate
"
metoo
and
"
unique"
in
vitro
assays.
The
following
paragraphs
summarize
the
Panel's
response
for
each
of
the
three
major
components
of
the
ICCVAM
performance
standards
for
in
vitro
tests.

Structural/
functional
components:

The
Panel
concurred
that
the
PS
prepared
by
ICCVAM
are
very
well
described
for
each
of
the
three
tests,
and
the
information
should
provide
a
basis
to
determine
whether
a
test
is
mechanistically
and
functionally
similar
to
a
validated
in
vitro
test
method.
The
Panel
stated
that
it
would
be
helpful
for
the
submitting
laboratories
if
the
Agency
provided
examples
of
what
they
would
consider
as
a
"
me­
too"
assay
or
a
new
assay,
based
upon
the
essential
structural
and
functional
elements
(
e.
g.,
human
skin
TER
vs.
rat
skin
TER).
There
was
some
concern
among
the
Panel
members
that
identification
of
a
"
me­
too"
10
of
22
assay
could
be
a
somewhat
subjective
process
rather
than
one
based
entirely
on
objective
criteria.
However,
with
the
limited
tests
that
have
been
evaluated
to
date
(
one
"
me­
too"
and
three
unique),
there
was
consensus
that
this
approach
of
using
structural
and
functional
equivalence
to
determine
a
"
me
too"
test
is
conceptually
feasible.

Reference
Chemicals:

The
Panel
recommended
that
NIEHS/
EPA
(
thru
ICCVAM)
develop
a
standard
list
of
reference
chemicals
for
validating
in
vitro
tests
and
establish
a
chemical
repository
for
reference
samples/
positive
controls
available
to
laboratories
for
developing/
conducting
in
vitro
skin
studies.
The
reference
panel
should
contain
sufficient
numbers
of
different
chemical
classes
(
with
a
range
of
potency,
solubility,
etc.)
to
establish
reasonable
performance
of
that
specific
test
for
those
particular
classes
of
chemicals.

For
the
three
validated
test
methods,
members
of
the
Panel
recommended
that
the
laboratories
be
allowed
to
determine
their
own
positive
control(
s)
and
suggested
that
the
PS
not
suggest
specific
examples
such
as
NaOH
pellets
and
10
N
HCl.
The
Panel
felt
that
these
particular
examples
may
be
too
corrosive,
and
if
suggested
by
the
Agency
as
a
positive
control,
could
become
the
"
gold
standard."
In
lieu
of
citing
specific
examples
for
positive
controls,
the
Panel
suggested
that
the
Agency
PS
provide
general
requirements
(
e.
g.,
well
characterized,
results
in
a
low­
to­
intermediate
response,
etc.)
wanted
in
a
positive
control
for
a
validated
test.

For
all
three
validated
tests,
the
Agency
PS
would
benefit
from
a
more
thorough
discussion
of
appropriate
benchmark
controls
(
range
of
severity,
classes
of
chemicals)
and
also
how
benchmark
controls
would
be
considered
in
the
validation
studies
of
the
assay.
The
Panel
also
recommends
that
minimum
replicate
requirements
be
specified
for
positive,
negative
and
benchmark
controls,
and
that
the
PS
be
unambiguously
stated.

Concordance
and
reliability
values:

The
Panel
suggested
that
the
Agency
provide
clear
guidance
on
requirements
necessary
to
establish
test
reliability
for
the
PS
for
each
validated
in
vitro
test
(
how
many
labs
for
the
interlaboratory
reliability
and
how
many
intra­
laboratory
replications?).
The
Panel
also
recommended
that
the
Agency
better
define
what
is
meant
by
comparable
concordance
for
test
accuracy
 
will
this
be
statistically
based?
The
Panel
expressed
the
view
that
the
PS
should
include
specific
guidelines
for
minimum
achieved
sensitivity
and
specificity
of
the
test
when
applied
to
the
reference
chemical
set.

One
Panel
member
expressed
the
view
that
if
there
is
no
appreciable
difference
in
performance,
an
in
vitro
assay
should
be
recommended
as
the
preferred
alternative
testing
method
for
use
over
an
ex­
vivo
assay
(
e.
g.,
rat
skin
TER)
as
the
former
more
directly
addresses
the
goal
of
animal
replacement.

Question
2
11
of
22
 
In
its
evaluation
of
any
mechanistically
similar
test
system,
the
Agency
plans
to
use
the
generic
criteria
used
by
ICCVAM
for
selecting
subsets
of
the
Reference
Chemicals
for
all
three
ICCVAM
Performance
Standards
documents.
The
criteria
specify
that
chemicals
should
be
selected
in
such
a
way
that
the
subset
includes
representatives
of
applicable
chemical
classes,
measures
a
range
of
corrosive
strengths,
includes
well­
defined
chemicals
that
are
currently
available
commercially,
and
has
unequivocal
animal
or
other
in
vivo
evidence.
Please
comment
on
the
strengths
or
weaknesses
of
this
approach
and
identify
and
discuss
any
modifications
to
the
criteria
that
should
be
considered.

Panel's
comments:

The
Panel
expressed
the
view
that
the
strength
of
the
PS
approach
to
validating
a
new
or
"
me
too"
in
vitro
test
derives
from
the
stated
selection
criteria
for
the
Reference
Chemical
set.
By
including
a
range
of
chemical
classes
in
the
Reference
Chemical
set
the
general
applicability
of
the
test
is
supported.
Choosing
Reference
Chemicals
exhibiting
a
broad
range
of
corrosive
strengths
provides
insight
into
the
quantitative
value
of
the
test.
This
could
be
important
for
assignment
of
corrosive
agents
to
packing
groups.
In
addition,
the
inclusion
of
mildly
corrosive
agents
supports
estimation
of
the
sensitivity
of
the
test.
The
use
of
well­
defined
agents
with
unequivocal
animal
or
other
in
vivo
evidence
in
regard
to
skin
corrosivity
anchors
the
Reference
Chemicals
as
valid
"
real
world"
representatives
and
allows
for
validated
comparisons
between
the
in
vitro
findings
and
the
potential
effects
of
actual
environmental
or
occupational
exposures.
Limiting
the
Reference
Chemical
set
to
commercially
available
chemicals
allows
for
the
widespread
use
of
this
testing
regimen.

The
Panel
identified
a
weakness
of
the
approach
in
that
it
may
be
difficult
to
include
a
sufficient
number
of
Reference
Chemicals
in
each
class,
both
corrosive
and
non­
corrosive,
which
meet
all
of
these
criteria.
The
Episkin/
Epiderm
Reference
Chemical
set
comes
closest,
with
6
of
8
classes
containing
both
corrosive
and
non­
corrosive
agents.
Although
numerous
classes
of
potentially
corrosive
chemicals
are
included
in
the
various
Reference
Chemical
sets,
some
classes
are
missing.
This
includes
inorganic
salts,
such
as
FeCl3,
which
was
reported
by
ECVAM
to
be
corrosive.
Also
the
Panel
noted
that
hydrocarbons
and
halogenated
hydrocarbons
are
common
solvents
and
diluents
for
pesticides,
and
that
these
chemicals
might
be
included
for
study
either
as
individual
agents
or
in
combination
with
other
chemicals.
The
question
of
how
many
"
classes"
the
test
methods
(
or
"
me­
too"
tests)
are
validated
with,
versus
the
number
of
classes
which
the
test
may
be
approved
for,
remains
unanswered.

The
Panel
pointed
out
that
a
second
weakness
of
the
PS
Reference
Chemical
descriptions
for
the
validated
in
vitro
tests
is
the
lack
of
standardization
of
the
list.
Different
groups
of
specific
chemical
agents
are
employed
(
or
recommended)
for
the
different
in
vitro
tests.
While
this
may
not
affect
the
validation
of
individual
test
systems,
it
does
impact
on
comparisons
between
the
available
and
proposed
test
systems.

Question
3
12
of
22
 
The
ICCVAM
approach
for
demonstrating
functional
similarity
of
"
me­
too"
test
methods
to
validated
methods
includes
the
use
of
well­
characterized
Reference
Chemicals
and
specifies
the
accuracy
and
reliability
that
should
be
achieved
by
"
me­
too"
test
systems
when
tested
in
intra­
and
inter­
laboratory
studies.
Please
comment
on
whether
"
me­
too"
test
systems
should
be
demonstrated
to
be
effective
for
evaluating
the
testing
endpoint
for
all
of
the
chemicals
in
the
Performance
Standard.
Please
comment
on
the
value
of
including
chemicals
with
range
of
potencies
in
the
Performance
Standard.
Under
what
circumstances
might
testing
of
"
me­
too"
systems
within
one
laboratory
ever
be
sufficient
to
demonstrate
functional
equivalence?

Panel's
comments:

The
Panel
agreed
that
a
minimum
number
of
Reference
Chemicals
(
subset
of
the
entire
list)
should
be
specified
in
the
PS,
to
be
used
for
validation
procedures
of
existing
alternative
test
methods,
as
well
as
"
me­
too"
tests.
It
was
noted,
for
example,
that
there
was
a
large
range
in
the
number
of
Reference
Chemicals
used
among
the
three
test
systems
presented,
with
a
low
of
12
to
a
high
of
122,
depending
upon
the
test
method
under
consideration.
Although
the
use
of
the
entire
original
Reference
Chemical
set
for
a
validated
test
method
for
validation
of
a
"
me­
too"
test
might
be
considered
excessive,
it
is
nonetheless
important
to
carry
out
a
sufficiently
broad
characterization
of
a
new
test
to
validate
its
performance.

The
approach
of
specifying
a
known
level
of
accuracy
and
reliability
for
a
"
me­
too"
test
to
be
considered
equivalent
to
the
validated
test
system
was
accepted
by
the
Panel.
Panel
members
suggested
that
Reference
Chemicals
be
limited
to
those
that
have
been
tested
with
sufficient
replication,
such
that
the
reliability
and
accuracy
estimates
themselves
are
considered
sufficiently
precise.
The
Panel
recommended
that
the
concordance
of
results
from
"
me­
too"
tests
be
established
by
comparison
to
the
unequivocal
properties
of
the
test
chemicals
in
human
or
animal
tests,
rather
than
by
comparison
to
an
alternative
test
method.
It
was
recognized
that
other
alternative
tests
may
have
less
than
100%
accuracy
(
sensitivity
and
specificity)
that
would
cloud
the
meaning
of
"
me­
too"
test
"
accuracy"
or
concordance.

One
Panel
member
considered
it
essential
that
if
as
few
as
12
chemicals
(
or
in
any
case
less
than
20)
are
specified
in
the
PS
(
as
was
the
EC's
specification
in
the
case
of
Corrositex
®
)
then
100%
concordance
with
in
vivo
test
results
should
be
required
to
demonstrate
test
equivalence
and
assure
the
public
safety.
Lower
percentage
concordance
would
be
acceptable
if
a
large
enough
subset
of
the
Reference
Chemicals
were
tested
so
as
to
include
more
than
one
chemical
from
all
classes
originally
validated,
with
a
range
of
potencies
or
responses
for
each
class.

While
recognizing
that
the
validated
test
provides
the
history
(
that
is,
the
empirical
criteria
for
acceptable
sensitivity,
selectivity,
etc.),
it
remains
questionable
whether
this
is
an
appropriate
"
bright­
line."
There
may
be
important
statistical
or
practical
considerations
to
the
choice
of
the
subset
of
Reference
Chemicals
to
be
included
in
the
PS.
One
Panel
member
queried
13
of
22
whether
the
decision
point
for
qualitative
judgment
of
corrosive
agents
is
sensitive
enough
to
detect
even
weakly
corrosive
agents,
stating
that
the
judgment
of
sensitivity
cannot
be
made
without
validation
using
known
weakly
corrosive
agents.
Thus,
the
"
Performance
Standard"
should
include:
1)
a
stated
minimum
number
of
diverse
test
chemicals,
from
all
relevant
chemical
classes;
(
2)
a
requirement
for
Reference
Chemicals
with
varying
potencies,
efficacies,
or
range
of
response,
ideally
within
each
chemical
class;
and
3)
minimum
standards
for
reliability
and
accuracy/
concordance
in
the
"
me­
too"
test
system
results
when
compared
to
the
known
properties
of
the
test
chemicals
for
in
vivo
tests.

A
majority
of
the
Panel
agreed
that
validation
of
a
"
me­
too"
test
in
a
single
laboratory
should
be
acceptable,
if
that
single
laboratory
is
the
only
practitioner
of
the
method.
The
criteria
for
acceptance
should
be
as
rigid
as
that
for
a
multi­
laboratory
validation.
This
would
involve
at
least
a
sufficient
number
of
independent,
repeated
tests
using
the
Reference
Chemicals
to
establish
the
concordance
of
the
"
me
too"
test
with
a
validated
test,
and
to
determine
the
intra­
laboratory
test
reliability
of
the
"
me
too"
test.

The
Panel
noted
the
importance
of
using
good
experimental
design
in
intra­
and
interlaboratory
studies,
being
concerned
that
there
was
little
discussion
of
batch­
to­
batch
(
or
peltto
pelt
in
the
case
of
TER)
variability
in
any
of
the
test
method
protocols,
data,
or
results.
The
implication
is
that
this
is
a
very
small
source
of
variability
for
these
test
systems,
which
may
not
be
the
case
in
future
systems.
The
general
procedures
for
evaluating
"
me­
too"
systems
should
take
this
into
account.

II.
Quality
Control
The
Agency
is
proposing
quality
control
measures
that
should
be
considered
when
evaluating
the
reliability
of
test
kits
for
regulatory
purposes.
Please
address
the
following
specific
issues.

Question
4
 
Subsets
of
the
Reference
Chemicals
used
in
test
method
validation
may
be
used
as
training
or
calibration
sets
by
testing
laboratories
using
in
vitro
systems.
Please
discuss
the
utility
of
and
necessity
for
training
or
calibration
sets
in
assuring
data
quality.
Please
comment
on
the
chemicals
selected
by
ICCVAM
for
use
as
a
calibration
set
for
TER
for
this
purpose.
Please
comment
on
the
ranges
of
chemical
classes
and
potencies
of
these
chemicals.
How
might
other
chemicals
be
selected
for
possible
use
in
the
calibration
sets?
Please
comment
on
the
value
of
identifying
chemicals
that
might
be
used
by
laboratories
as
training
sets
to
demonstrate
proficiency
in
performing
the
test.

Panel's
comments:
14
of
22
Given
the
nature
of
these
in
vitro
systems,
particularly
in
regard
to
lot­
to­
lot
and
day­
to­
day
variability,
the
Panel
felt
it
essential
that
test
system
performance
be
established
and
understood.
A
simple
positive
and
negative
control
may
not
be
sufficient
to
represent
the
range
of
responses
and
the
sensitivity
required
for
detection
of
weakly
corrosive
agents.
In
the
case
of
the
Corrositex
®
test
the
twelve
Reference
Chemicals
suggested
by
ICCVAM
meet
the
criterion
of
including
strongly
and
weakly
corrosive
and
non­
corrosive
agents.
However,
12
chemicals
constitute
a
limited
test
set.
It
also
is
incomplete;
missing
are
potentially
corrosive
inorganic
salts
like
Fe(
Cl)
3,
which
is
noted
in
the
60
chemical
ECVAM
list.
Further,
the
ECVAM
"
60"
list
does
not
completely
reflect
the
classes
of
chemicals
that
are
important
with
regard
to
pesticide
registration.
Hydrocarbon
solvents,
for
example,
find
use
as
diluents
but
are
not
included
in
the
list.
While
most
of
these
solvents
are
complex
mixtures,
toxicity
profiles
can
be
established
both
for
the
mixture
and
for
suitable
single­
chemical
surrogates
(
e.
g.,
toluene,
decane,
etc).
Clearly
a
balance
must
be
struck
between
maintaining
a
manageable
number
of
Reference
Chemicals
and
assuring
that
all
relevant
mechanistic
and
chemical
classes
are
included.

While
the
background
documents
discuss
the
need
for
a
range
of
potencies
for
chemicals,
it
is
important
that
Reference
Chemicals
that
represent
a
range
of
implementation
difficulties
be
included
as
well.
Part
of
the
calibration
process
for
testing
laboratories
is
that
the
technicians
learn
to
be
consistent
in
application
so
that
reproducible
results
will
be
obtained
for
the
Reference
Chemicals
over
time.
The
potency
of
a
chemical
may
not
be
the
best
measure
of
how
difficult
it
is
for
a
technician
to
get
consistent
results
with
that
chemical.
The
Reference
Chemical
set
should
include
some
chemicals
that
are
difficult
to
work
with,
thereby
challenging
the
technical
skill
of
the
staff
and
forcing
them
to
"
stay
skilled."
Further,
some
chemicals
(
e.
g.,
solvents)
may
destroy
the
test
system;
knowledge
of
this
is
important
if
such
a
chemical
is
tested
in
a
formulated
product.

The
Panel
noted
that
training
in
the
use
of
the
validated
test
is
required
to
be
documented
under
GLPs,
presumably
with
Reference
Chemicals.
One
panel
member
expressed
caution
regarding
the
use
of
the
terms
proficiency
and
calibration
set.
Proficiency
implies
a
precision
and
accuracy
as
may
be
required
by
independent
accreditation.
The
training
to
meet
this
objective
is
a
laboratory
management
function.
The
term,
"
calibration
set"
implies
traceability
to
some
standard,
e.
g.,
a
national
standard.
In
the
context
of
the
Panel's
discussion,
Reference
Chemicals
are
identified
that
can
be
used
as
control
or
benchmark
chemicals
to
help
standardize
or
validate
a
method
in
a
laboratory
and
monitor
its
performance
but
may
not,
in
the
strictest
sense,
be
a
true
calibration
of
the
test
results.

For
a
training
set
of
chemicals
to
be
used
either
initially
or
at
some
set
intervals
for
the
validation
of
an
assay
and
its
performance
in
a
given
laboratory,
this
balance
between
number
of
chemicals
and
inclusivity
shifts
to
a
higher
number
of
individual
chemicals.
Whereas
twelve
might
be
an
appropriate
number
for
regular
"
calibration"
a
training
and
validation
set
could
easily
be
2­
3
times
this
number.
This
would
ensure
coverage
of
relevant
classes
and
potencies
for
corrosive
agents
and
better
test
the
abilities
of
a
given
laboratory
to
perform
the
assay
accurately.
15
of
22
The
Panel
expressed
the
view
that
Reference
Chemical
testing:

 
Provides
relevant
training
and
documentation
of
training
as
required
by
GLPs;
 
Provides
a
means
to
evaluate
technician
competency
for
the
test
method;
 
Permits
comparison
to
a
validation
database
and
assessment
of
variability
among
labs;
 
Identifies
relative
strengths/
weaknesses
of
the
lab
and
whether
additional
training
is
needed.

Question
5
 
Anticipating
the
use
of
systems
using
tissue
constructs,
ex
vivo
systems,
microarrays
or
genetically
modified
cells,
please
discuss
aspects
of
the
quality
control
criteria
that
are
necessary
for
assuring
the
integrity
of
such
systems
over
time
and
from
lot­
to­
lot.
Please
comment
on
whether
and
how
the
type
of
system
­
tissue
constructs,
ex
vivo
systems,
or
genetically
modified
cells
or
animals
­
should
affect
the
criteria
for
quality
control
for
assuring
the
integrity
of
such
systems,
both
over
time
and
from
lot­
to­
lot.

Panel's
comments:

The
use
of
PS,
positive
controls,
negative
controls
and
benchmark
controls
will
provide
the
opportunity
to
achieve
a
degree
of
control
over
the
quality
of
Proprietary
Test
Methods
(
PTMs).
Two
issues
that
have
not
been
addressed
in
the
PS
are
how
drift
in
the
PTMs
will
be
monitored
and
how
information
about
problems
that
arise
from
the
use
of
these
controls
will
be
assimilated
and
evaluated
by
the
vendor.
Individual
test
facilities
may
detect
failures
or
out­
of­
specification
performance
of
the
PTM
and
proceed
according
to
their
operating
procedures,
but
the
lack
of
GMP­
like
regulatory
authority
does
not
require
these
failures
to
be
reported
to
and
addressed
by
the
vendor.

Other
facilities
may
then
use
an
inadequate/
under­
performing
PTM
or
lot
of
PTM
without
benefit
of
the
experiences
of
the
first
facility.
There
should
be
some
consideration
that
PTM
performance
reports
be
compiled
by
the
vendor
and
reported
to
purchasers
of
the
PTM.
Similar
mechanisms
are
used
by
computer
software
vendors
to
alert
purchasers
of
their
products
of
problems
or
issues
with
their
products.

The
answer
to
the
second
part
of
this
question
goes
beyond
the
immediate
concerns
of
the
Panel,
which
were
in
vitro
tests
for
corrosive
chemicals.
Rather,
the
answer
discusses
general
considerations
for
future
in
vitro
tests
that
will
incorporate
the
newest
advances
that
are
being
made
in
molecular
biology.
All
testing
systems
require
quality
control
for
assuring
reproducibility,
sensitivity,
and
specificity.
Otherwise,
results
from
the
same
test
repeated
in
the
same
laboratory,
or
in
different
laboratories,
could
not
be
compared.
Incorporating
positive
and
negative
controls,
as
well
as
benchmark
samples,
monitors
quality
control.
The
specific
types
of
controls,
the
number
of
controls,
the
frequency
of
inserting
these
controls
16
of
22
and
the
benchmark
samples,
however,
will
likely
be
different
for
different
types
of
assays.
The
number
of
controls
would
be
expected
to
increase
in
highly
variable
systems
(
e.
g.
those
that
require
animals)
but
must
be
limited
because
of
cost
considerations.
Hence,
the
development
of
newer
testing
systems
that
limit
variability
would
have
substantial
benefit.

An
example
of
the
concern
for
variability
is
an
ex
vivo
system,
in
which
tissue
is
excised
from
a
donor
and
cultured
as
either
organ
culture,
explants,
or
dissociated
cells.
There
will
be
variability
in
each
type
of
culture
because
of
variability
in
the
donor
animals.
In
primary
cultures,
however,
the
variability
can
be
greatly
limited
if
large
batches
of
cells
are
prepared
from
several
animals
and
frozen.
New
testing
systems
for
screening
different
types
of
toxic
chemicals
will
likely
be
developed
using
genetically
modified
cell
lines.
The
Ames
assay
is
one
example
of
an
already
established
test
that
uses
genetically
modified
bacteria
to
screen
for
mutagens,
which
are
possible
carcinogens.
A
more
complex
test
system
could
be
developed
to
establish
tissue
constructs.
For
example,
a
testing
system
might
be
developed
that
uses
genetically
modified
skin
stem
cell
lines
that
differentiate
into
skin.
The
currently
available
tissue
construct
uses
skin
epithelial
cells
from
donors.
The
advantage
of
the
stem
cell
line
is
that
the
lot­
to­
lot
variability
would
be
reduced
because
the
source
of
variability,
donor
tissue,
would
be
reduced.

Microarrays
(
gene
arrays)
are
powerful
endpoint
assays
that
measure
changes
in
the
expression
of
hundreds
or
thousands
of
genes
and
will
likely
be
used
in
different
types
of
testing
systems.
One
such
use
would
be
in
classifying
xenobiotics
according
to
the
patterns
of
genes
that
they
induce.
The
pattern
of
gene
expression
has
been
termed
a
gene
fingerprint,
and
testing
systems
might
be
developed
for
screening
xenobiotics
by
measuring
gene
fingerprints.
In
measuring
gene
fingerprints,
rather
than
one
or
two
specific
genes,
the
testing
system
has
more
power
for
statistical
analysis
and
will
likely
produce
more
consistent
data.
Gene
arrays
also
have
the
potential
of
reducing
the
number
of
required
controls.
For
example,
testing
systems
for
determining
gene
fingerprints
for
xenobiotics
must
use
cell
lines
that
express
enzymes
that
metabolize
xenobiotics.
Positive
controls
should
be
incorporated
in
the
testing
systems
for
screening
xenobiotics
to
validate
the
presence
of
these
enzymes.
In
using
gene
arrays,
the
positive
controls
might
not
be
necessary
because
the
expression
of
the
activating
enzymes,
as
well
as
the
gene
fingerprints,
would
be
determined
in
the
same
gene
array.

The
Panel
noted
that
microarrays
and
other
related
systems
seem
to
have
a
long
way
to
go
toward
producing
reproducible
responses
among
true
replicates.
In
fact,
very
little
true
replication
is
being
done,
primarily
due
to
the
expense
of
each
replicate.
As
the
state
of
the
art
in
microarray
use
becomes
mature,
true
replication
with
demonstrated
repeatability
may
become
the
standard.
When
that
is
truly
the
case,
test
systems
based
on
this
technology
should
provide
useful
tools
for
risk
evaluations.
As
these
new
tests
are
put
into
practice,
more
attention
must
be
focused
on
how
drift
in
performance
test
standards
will
be
monitored
and
how
information
about
these
problems
will
be
assimilated
and
evaluated
by
the
vendor.

Question
6
17
of
22
 
Please
comment
on
the
advantages
and
disadvantages
of
including
concurrent
positive
and
negative
controls
with
in
vitro
assays
when
used
as
alternatives
to
animal
testing.
What
are
the
important
characteristics
of
positive
and
negative
controls
for
in
vitro
studies?
What
aspects
of
positive
control
characteristics
allow
them
to
be
used
as
part
of
the
quality
control
process?
When
might
confirmation
that
positive
controls
are
performing
within
expected
or
historical
limits
be
sufficient
to
demonstrate
that
the
Proprietary
Test
Method
or
non­
proprietary
assay
system
is
functioning
properly?
When
might
additional
quality
control
measures
be
needed?

Panel's
comments:

The
Panel
commented
that
insufficient
controls
may
preclude
meaningful
interpretation
of
in
vitro
test
results.
Despite
the
fact
that
positive
and
negative
controls
are
not
often
used
in
in
vivo
studies,
they
are
routinely
included
in
in
vitro
studies
and
it
is
clearly
advantageous
and
desirable
that
they
be
used
in
the
test
systems
being
discussed
here.
Positive
and
negative
(
and
vehicle)
controls
provide
needed
checks
within
a
study
that
tell
the
investigator
that
the
test
system
appears
to
be
intact
and
functional.
Positive
controls
help
identify
performance
variability
between
technicians,
between
laboratories
and
between
lots
of
test
system.
Appropriate
controls
will
likely
be
needed
for
some
length
of
time
until
the
Agency
and
practitioners
are
satisfied
with
the
performance
of
the
test
over
time
and
across
laboratories.
From
a
quality
control
perspective,
temporal
monitoring
of
controls
across
studies
and
laboratories
will
help
establish
consistency
of
response
for
the
test
system.

The
Panel
again
pointed
out
that
a
single
positive
control
per
assay
may
not
be
sufficient
and
that
it
may
be
desirable
to
include
positive
control
chemicals
for
one
or
more
of
the
classification
severities.
At
least
one
Panel
member
queried
as
to
what
actions
should
be
taken
when
a
negative
control
produces
a
positive
response
or
a
positive
control
produces
a
negative
response.
The
answer
will
depend
on
the
degree
of
replication
assigned
to
controls
and
the
specified
minimum
accuracy
or
concordance
for
the
test
system.
Clearly,
the
developers
of
the
test
system
should
incorporate
into
the
recommended
protocols
guidance
on
the
degree
of
replication
needed
for
controls,
and
what
actions
should
be
taken
when
unexpected
results
are
observed
with
controls.
The
degree
of
replication
should
be
based
upon
the
expected
variability
and
the
levels
of
specificity
and
sensitivity
displayed
by
the
test
systems
for
the
Reference
Chemicals
used
as
controls.
With
adequate
replication,
the
fact
that
positive
controls
(
and
negative
and
vehicle
controls,
for
that
matter)
are
performing
within
expected
limits
should
be
sufficient
to
demonstrate
that
the
test
system
is
functioning
properly.

Question
7
 
Does
the
Panel
agree
that
the
benchmark
controls
serve
a
useful
purpose
to
demonstrate
18
of
22
the
level
of
response
that
can
be
expected
for
each
chemical
class
for
each
lot
of
Proprietary
Test
Method
assays?
Can
the
Panel
suggest
criteria
for
choice
of
appropriate
benchmark
controls?

Panel's
comments:

The
Panel
agrees
that
benchmark
controls
are
an
important
mechanism
to
assess
both
the
adequacy
of
the
method
as
well
as
lot­
to­
lot
variability
and
should
be
considered
as
a
standard
component
of
these
test
methods.
Benchmark
controls,
as
well
as
positive
and
negative
controls,
should
be
tested
in
each
new
lot
to
determine
the
viability
and
usability
of
each
lot.
Control
charts
could
assess
variability
among
lots,
and
provide
a
basis
for
acceptance/
rejection.
The
Panel
suggests
that
benchmark
controls
include
several
"
classic"
responders
from
different
chemical
classes/
mode
of
actions.

Variability
between
different
lots
is
a
major
concern
of
the
Panel
and
must
be
assessed
with
negative
and
positive
controls
as
well
as
benchmark
samples.
The
number
of
controls
and
samples
depends
on
several
factors;
many
of
which
will
be
defined
by
the
specific
test.
Certain
tests
are
very
consistent
and
require
fewer
positive
controls
and
benchmark
samples
for
assessing
lot­
to­
variability
whereas
other
tests
are
less
consistent.
The
Panel
agreed
that
there
is
concern
regarding
whether
the
lots
are
large
enough
to
accommodate
these
types
of
controls.
To
address
this
concern,
the
Panel
suggests
that
EPA
establish
the
necessary
controls
and
benchmark
samples
in
the
individual
tests
and
consults
with
the
manufacturer
of
the
test.
Accordingly,
the
manufacturer
would
be
encouraged
to
change
production
so
that
the
size
of
lots
are
sufficient
for
allowing
adequate
controls
and
benchmarks.

Appropriate
benchmark
controls
should
have
the
following
properties:

 
Consistent
and
reliable
source(
s)
for
the
chemical
 
Structural
and
functional
similarity
to
the
class
of
article
being
tested
 
Known
physical/
chemical
characteristics
 
Supporting
data
on
known
effects
in
animal
models
 
Known
potency
in
the
range
of
response
(
including
moderate
response)

One
Panel
member
stated
that
benchmark
controls
can
serve
a
very
useful
purpose,
especially
in
the
situation
where
the
test
system
demonstrates
significant
batch­
to­
batch
variability
in
response.
But
this
variability
has
not
been
directly
addressed
for
the
test
systems
being
discussed
here.
If
we
assume
that
such
variability
is
quite
low,
the
benefit
of
re­
running
benchmark
controls
for
each
batch
is
reduced.
In
this
case,
the
use
of
benchmark
controls
might
be
relegated
to
a
supplier
QC
role
with
periodic
running
of
benchmark
chemical
to
ensure
continued
consistency
of
response
over
time.
On
the
other
hand,
if
the
test
system
does
demonstrate
significant
batch­
to­
batch
variability,
it
would
be
important
to
run
benchmark
controls
more
often.
Finally,
it
would
seem
that
benchmark
controls
would
be
more
important
in
calibrating
a
formal
dose
response
model.
The
need
for
these
controls
then
depends
on
the
level
of
precision
needed
in
the
final
model.
19
of
22
20
of
22
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2003.

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