Page
1
of
13
October
2,
2003
BACKGROUND
FOR
FIFRA
SCIENTIFIC
ADVISORY
PANEL
ON
REGULATORY
USE
OF
IN
VITRO
SYSTEMS
Executive
Summary
Many
in
vitro
and
ex
vivo
methods
have
been
developed
or
are
under
development
to
reduce
or
replace
animal
usage
in
toxicity
tests.
Across
United
States
federal
agencies,
the
Interagency
Coordinating
Committee
for
Validation
of
Alternative
Methods
(
ICCVAM)
typically
provides
for
review
and
assessment
of
the
status
of
validity
of
test
systems,
including
Proprietary
Test
Methods
(
Refs.
1,
2).
Thereafter,
the
Environmental
Protection
Agency
determines
if
data
generated
using
the
new
methods
are
acceptable
for
its
regulatory
mandate.
A
process
is
needed
to
provide
assurance
that
an
in
vitro
system
continues
to
perform
over
time
in
a
manner
that
is
consistent
with
the
test
system
as
it
was
originally
validated.
If
a
validated
Proprietary
Test
Method
is
of
interest
to
the
Agency
for
regulatory
testing,
the
Agency
writes
a
generic
test
guideline
which
spells
out
Performance
Standards
that
must
be
met
by
any
specific
test
system
falling
under
it.
Consistent
with
its
goal
of
obtaining
scientifically
sound
test
data
for
hazard
and
risk
assessment
of
pesticides
and
toxic
chemicals,
the
Agency
is
exploring
what
changes
in
current
policies
and
procedures
may
be
needed
to
facilitate
the
acceptance
of
data
developed
using
in
vitro
alternatives.

The
Agency
is
working
with
national
and
international
organizations
to
encourage
development
of
policies
and
"
standards"
for
scientific
practice
to
assure
quality
in
implementation
of
in
vitro
methods
performed
as
alternatives
to
animal
testing
for
regulatory
purposes.

The
Agency
will
draw
on
Performance
Standards
when
writing
generic
guidelines
for
Proprietary
Test
Methods;
identify
quality
control
measures
for
in
vitro
methods
in
Office
of
Prevention,
Pesticides,
and
Toxic
Substances
test
guidelines
(
to
the
extent
feasible
­
since
companies
may
declare
some
quality
control
measures
to
be
confidential);
and
identify
appropriate
controls,
data
reporting
elements,
and
benchmarks
in
test
guidelines
so
that
the
potential
risks
of
pesticides
and
other
chemicals
can
be
reviewed
and
reliably
assessed.

The
Office
of
Prevention,
Pesticides,
and
Toxic
Substances
intends
to
draw
on
existing
Good
Laboratory
Practice
regulations
to
ensure
that
in
vitro
tests
used
for
regulatory
purposes
are
reproducible,
credible,
and
acceptable.
Manufacturers
and
testing
laboratories
will
be
able
to
refer
to
Office
of
Prevention,
Pesticides,
and
Toxic
Substances
test
guidelines
and
ICCVAM
essential
test
method
components
specified
by
ICCVAM
for
quality
control
measures
for
use
under
Good
Laboratory
Practice
regulations.
In
addition,
the
Agency
is
considering
the
utility
of
technical
guidance
to
use
when
laboratories
performing
in
vitro
studies
as
alternatives
to
animal
use
for
regulatory
purposes
are
audited
under
Good
Laboratory
Practice.
Page
2
of
13
BACKGROUND
FOR
FIFRA
SCIENTIFIC
ADVISORY
PANEL
ON
REGULATORY
USE
OF
IN
VITRO
SYSTEMS
I.
INTRODUCTION:
REGULATORY
USE
OF
IN
VITRO
ALTERNATIVE
TEST
METHODS
Background:

Historically,
in
vivo
tests
in
laboratory
animals
have
formed
the
foundation
of
hazard
and
risk
assessment
at
the
Environmental
Protection
Agency
(
EPA).
The
development
of
sound
testing
procedures
for
good
science
in
regulation
includes
incorporation
of
the
latest
scientific
advances,
including,
whenever
practical,
validated
and
accepted
non­
animal
methods
such
as
cell
and
tissue
cultures,
bioconstructs
or
microarrays,
as
testing
alternatives
to
animal
testing.

Many
such
in
vitro
methods
have
been
developed
or
are
under
development
to
reduce
or
replace
animal
usage
in
toxicity
tests.
Any
ex
vivo
tissue,
bioconstruct
or
cultured
cell
has
a
limited
number
of
ways
to
respond
to
a
chemical
stimulus,
whereas
intact
animals
have
many
other
potentialities.
Consistent
with
its
goal
of
obtaining
scientifically
sound
test
data
for
hazard
and
risk
assessment
of
pesticides
and
toxic
chemicals,
the
Agency
is
exploring
what
changes
in
current
policies
and
procedures
may
be
needed
to
facilitate
the
acceptance
and
use
of
data
developed
using
in
vitro
alternatives.
Some
in
vitro
methods
are
developed
by
sponsors
for
commercial
marketing
as
Proprietary
Test
Methods
(
PTMs).
Some
components
and
principles
of
such
tests
may
not
always
be
divulged
to
the
public.
In
other
cases,
sponsors
may
develop
in
vitro
methods
and
disclose
their
test
design
and
the
scientific
principles
of
the
test;
these
may
or
may
not
be
PTMs.

Normally,
when
new
in
vitro
test
methods
are
developed,
systematic
laboratory
studies
are
performed
with
a
set
of
Reference
Chemicals.
These
chemicals
should
illustrate
the
range
of
responses
of
the
test
system
and
be
representative
of
the
chemical
classes
for
which
the
test
is
expected
to
be
used.
The
chemicals
are
tested
in
the
new
in
vitro
test
and
compared
to
the
existing
standard
or
traditional
animal
test
or
human
data
and
experience.
Optimally,
there
should
also
be
in
vitro
results
from
testing
of
the
same
materials
in
several
different
laboratories
for
both
the
in
vitro
and
the
in
vivo
test
method
(
Ref.
1).
Validation
assesses
the
new
test
system's
ability
to
predict
the
intact
organism
for
the
toxicological
effect
of
interest.
In
the
course
of
its
validation,
the
test's
reliability
is
assessed
in
terms
of
intra­
and
inter­
laboratory
variability.
Across
United
States
federal
agencies,
the
Interagency
Coordinating
Committee
for
Validation
of
Alternative
Methods
(
ICCVAM)
typically
provides
for
review
and
assessment
of
the
status
of
validity
of
test
systems,
including
PTMs
(
Refs.
1,2).
Thereafter,
the
Agency
determines
if
data
generated
using
the
new
methods
are
acceptable
for
its
regulatory
mandate.
Page
3
of
13
Quality
and
Consistency:

In
vitro
alternatives
to
animal
testing
pose
unique
issues
regarding
quality
and
performance.
Such
in
vitro
systems
also
must
be
maintained
appropriately
to
ensure
their
quality
and
integrity,
e.
g.,
sterility,
viability,
and
responsiveness.
Therefore,
a
process
is
needed
to
provide
assurance
that
an
in
vitro
system
continues
to
perform
over
time
in
a
manner
that
is
consistent
with
that
of
the
test
system
as
it
was
originally
validated.
Consistency
of
performance
of
the
in
vitro
system
is
also
needed
with
any
change
in
ingredients
or
the
manufacturing
practices
for
proprietary
test
systems,
or
procedural
variations
among
the
laboratories
performing
the
test.

Performance
Standards:

Under
law,
United
States
agencies
are
limited
in
endorsement
of
validated
PTMs
unless
generic
guidelines
and
Performance
Standards
are
developed
for
each
method
(
Ref.
3).
Performance
Standards
include
a
description
of
the
essential
structural
and
procedural
elements
of
the
test
method
and
the
levels
of
accuracy
and
reliability
that
the
test
method
should
achieve
when
evaluated
using
chemicals
selected
from
among
the
Reference
Chemicals
which
were
used
to
demonstrate
the
acceptable
performance
of
the
validated
PTM.
In
addition,
Performance
Standards
for
each
in
vitro
test
system
can
be
used
to
ensure
that
the
assay
system
and
the
laboratory
are
performing
as
expected
and
in
accordance
with
performance
of
the
in
vitro
system
as
originally
validated.

Quality
Framework
for
Test
Data
Submitted
to
the
Agency:

Quality
control
requirements
for
assays
performed
to
fulfill
regulatory
requirements
are
included
in
the
Agency's
Good
Laboratory
Practice
regulations
(
GLP)(
Ref.
4).
The
Office
of
Pesticide
Programs
intends
to
draw
on
currently
promulgated
GLP
regulations
as
well
as
good
scientific
practices
established
by
various
scientific
disciplines
relevant
to
in
vitro
systems
to
ensure
that
in
vitro
tests
used
for
regulatory
purposes
are
reproducible,
reliable,
credible
and
acceptable.
The
Agency
is
also
considering
developing
special
guidance
for
its
quality
control
auditors
to
use
with
in
vitro
tests
submitted
for
regulatory
purposes
The
Office
of
Prevention,
Pesticides,
and
Toxic
Substances
(
OPPTS)
has
long
standing
practices
for
quality
assurance
of
test
data
submitted
to
the
Agency
for
use
under
the
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(
FIFRA).
The
Office
of
Pesticide
Programs
generally
bases
it
regulatory
decisions
under
FIFRA
and
the
Food,
Drug
and
Cosmetics
Act
on
guideline
studies
which
conform
to
GLP
regulations.
OPPTS
test
guidelines
spell
out
important
quality
control
and
data
reporting
elements.
These
must
be
documented
for
thorough
review
by
the
Agency.
The
Office
of
Pesticide
Programs
has
also
specified
acceptance
criteria
for
use
with
studies
which
might
have
preceded
GLP
or
may
have
been
performed
for
other
regulatory
authorities.
The
Agency
has
a
comprehensive
quality
assurance
program
to
ensure
the
quality
of
data
which
supports
our
regulatory
decisions
(
Ref.
5).
OPPTS
has
historically
acted
consistently
with
the
Page
4
of
13
thrust
of
this
program
due
to
its
longstanding
use
of
quality
assurance
practices.

Anticipating
the
use
of
in
vitro
methods
as
alternatives
to
animal
studies,
the
Agency
plans
to
standardize
data
reporting
elements
for
cell
cultures,
microarrays,
and
other
in
vitro
or
in
silico
methods.
Efforts
are
already
in
place
in
other
fora
to
identify
appropriate
data
elements
for
such
studies,
e.
g.,
the
report
of
European
Center
for
Validation
of
Alternative
Methods
(
ECVAM)
Good
Cell
Culture
Practice
Task
Force
(
Ref.
6)
;
and
the
Minimum
Information
About
a
Microarray
Experiment
­
MIAME
for
Toxicogenomics
(
MIAME­
Tox)
report
by
the
Microarray
Gene
Expression
Data
Society
(
Ref.
7).
The
Agency
is
following
this
work
and
will
use
such
efforts
as
a
starting
point
for
developing
its
own
policies.
In
addition
to
identifying
data
reporting
elements,
the
Office
of
Prevention,
Pesticides
and
Toxic
Substances
will
identify
all
appropriate
quality
control
steps
for
laboratories
to
implement
when
using
in
vitro
tests
as
alternatives
to
animal
testing.

II.
PERFORMANCE
STANDARDS
Proprietary
Test
Methods:

United
States
agencies
may
identify
proprietary
in
vitro
or
ex
vivo
test
methods
in
its
test
guidelines
if
they
comply
with
the
Performance
Standards.
If
a
validated
PTM
is
of
interest
to
the
Agency
for
regulatory
testing,
the
Agency
writes
a
generic
test
guideline
which
spells
out
Performance
Standards
that
must
be
met
by
any
specific
test
system
falling
under
it.
Performance
Standards
can
be
used
in
two
ways.
Firstly,
they
are
applicable
to
the
specific
validated
PTM,
allowing
that
test
system
to
be
used
for
regulatory
purposes.
Secondly,
the
Performance
Standards
identify
criteria
that
should
be
met
by
any
other
future
PTM
that
would
fall
under
the
generic
test
guideline.
This
means
that
any
other
PTM
that
meets
the
Performance
Standards
can
be
identified
as
such.

ICCVAM
has
described
a
process
and
developed
definitions
addressing
ways
to
handle
validation
of
PTMs,
including
setting
Performance
Standards
(
Ref.
2).
Performance
Standards,
based
on
a
validated
test
method,
provide
a
basis
for
evaluating
the
comparability
of
a
proposed
test
method
that
is
mechanistically­
and
functionally­
similar,
i.
e.
a
"
me­
too"
test
method.
The
three
elements
of
Performance
Standards
are
(
a)
the
essential
test
method
components
of
the
validated
test
method,
(
b)
a
list
of
recommended
Reference
Chemicals
drawn
from
the
test
systems's
validation
data
base
and
(
c)
a
statistical
description
of
the
accuracy
and
reliability
that
should
be
achieved
by
the
proposed
test
system
when
applied
to
the
subset
of
Reference
Chemicals.
Essential
test
method
components
are
descriptive
structural,
functional,
and
procedural
elements
of
a
validated
test
method
that
should
be
included
in
the
protocol
of
a
proposed,
me­
too
test
method.
These
include
unique
characteristics
of
the
test
method,
critical
procedural
details,
and
certain
quality
control
measures.
Adherence
to
essential
test
method
components
will
help
to
assure
that
a
proposed
test
method
is
based
on
the
same
concepts
as
the
corresponding
validated
test
method.
The
unique
characteristics
of
in
vitro
alternative
test
systems
generally
address
the
target
tissue
and
the
detection
method.
Critical
procedural
details
include
mode
of
chemical
Page
5
of
13
application,
controls,
and
reporting
elements.

After
a
test
method
has
been
accepted
as
valid
for
its
purpose
in
fulfilling
data
requirements
for
registration
or
reregistration
under
FIFRA,
a
representative
subset
of
chemicals
used
during
the
validation
process
may
be
selected
to
validate
"
me­
too"
test
methods.
To
the
extent
possible,
this
subset
of
Reference
Chemicals
should:


Be
representative
of
the
range
of
responses
that
the
validated
test
method
is
capable
of
measuring
or
predicting

Have
produced
consistent
results
in
the
validated
test
method
and
in
the
reference
test
method
and/
or
the
species
of
interest

Reflect
the
accuracy
of
the
validated
test
method

Have
well­
defined
chemical
structures

Be
readily
available

Not
be
associated
with
excessive
hazard
or
prohibitive
disposal
costs
This
subset
of
Reference
Chemicals
represents
the
minimum
number
of
chemicals
that
should
be
used
to
evaluate
the
performance
of
a
proposed,
"
me­
too"
test
method
.
They
allow
the
performance,
in
terms
of
accuracy,
sensitivity,
specificity,
false
positive
rates,
and
false
negative
rates,
of
the
test
method
for
relevant
chemical
classes
to
be
determined.
Here,
accuracy
means
(
a)
The
closeness
of
agreement
between
a
test
result
and
an
accepted
reference
value
and
is
measured
as
the
proportion
of
the
correct
outcomes
of
the
methods;
sensitivity
is
the
proportion
of
all
positive
test
materials
that
are
correctly
classified
as
positive
in
a
test;
specificity
is
the
proportion
of
all
negative
test
materials
that
are
correctly
classified
as
negative
in
a
test;
false
positive
rate
is
the
proportion
of
all
negative
test
materials
that
are
falsely
identified
as
positive
and;
false
negative
rate
is
the
proportion
of
all
positive
test
materials
that
are
falsely
identified
as
negative.

A
"
me­
too"
test
system
may
be
manufactured
or
marketed
by
a
different
company
from
the
original
test
system
sponsor.
The
"
me­
too"
system
is
mechanistically
and
functionally
similar
to
the
original
validated
system,
and
is
not
just
intended
to
be
a
different
system
for
measuring
the
same
endpoint.
For
example,
two
test
systems
using
human
skin
models
for
assessing
dermal
corrosion,
discussed
in
the
section
below,
are
EPISKINTM
and
EpiDermTM
.
EpiDerm
is
a
"
me­
too"
test
method
for
EPISKIN
and
was
validated
against
a
subset
of
the
Reference
Chemicals
used
to
validate
EPISKIN
(
Ref.
8).
Manufacturers
may
use
essential
test
method
components
to
demonstrate
that
the
"
me­
too"
method
is
mechanistically
similar
to
a
validated
Page
6
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13
PTM.
Comparable
performance
of
the
"
me­
too"
method
for
the
subset
of
Reference
Chemicals
can
demonstrate
its
functional
similarity
without
the
need
for
a
full
validation.

Performance
Standards
required
for
PTMs
approved
for
use
under
FIFRA
can
also
be
part
of
the
foundation
for
quality
control
of
PTMs.
(
See
section
III).
Performance
Standards
should
be
designed
to
allow
manufacturers
or
applicants
to
demonstrate
to
the
Agency
that
the
test
kit
as
marketed,
whether
a
"
me­
too"
or
a
PTM
product,
performs
as
scientifically
validated.

Much
of
the
activity
to
develop
non­
animal
methods
for
toxicological
evaluation
has
been
and
is
expected
to
be
at
the
behest
of
commercial
sponsors.
United
States
agencies
need
Performance
Standards
for
all
new
PTMs.
Use
of
Performance
Standards
is
also
desirable
for
other
in
vitro
methods
as
well.
Availability
of
Performance
Standards
can
be
expected
to
facilitate
the
development
of
"
me­
too"
test
methods
while
setting
standards
for
the
accuracy
and
reliability
of
the
original
test
method
and
any
mechanistically
and
functionally
similar
methods.

Performance
Standards
for
TER,
Corrositex,
and
EPISKIN/
EpiDerm:

ICCVAM
previously
evaluated
and
recommended
four
validated
test
methods
for
assessing
the
dermal
corrosivity
hazard
potential
of
chemicals:
Corrositex
®
,
EPISKIN
TM,
EpiDerm
TM
(
EPI­
200),
and
the
rat
skin
transcutaneous
electrical
resistance
(
TER
Assay
(
Refs.
8,9).
Corrositex
uses
a
biological
gel
supported
by
an
inert
semipermeable
membrane
to
determine
the
ability
of
a
corrosive
chemical
or
mixture
to
pass
through,
by
diffusion
and/
or
destruction/
erosion
and
elicit
a
color
change
in
an
underlying
liquid
indicator.
EPISKIN
uses
a
three­
dimensional
human
skin
model
composed
of
human
collagen
and
human
keratinocytes
and
utilizes
cell
viability
as
the
measured
endpoint.
EpiDerm
is
mechanistically
and
functionally
related
to
EPISKIN
and
consists
of
a
three­
dimensional
human
skin
model
utilizing
cultured
epidermal
keratinocytes,
with
cell
viability
being
the
endpoint.
The
TER
assay
uses
disks
of
ex
vivo
rat
skin
to
which
the
application
of
corrosive
material
produces
a
loss
of
normal
stratum
corneum
integrity
and
barrier
function
as
measured
by
a
reduction
of
the
inherent
transcutaneous
electrical
resistance
below
a
predetermined
threshold
level.
The
Agency
requested
that
ICCVAM
establish
Performance
Standards
for
the
three
proprietary
dermal
corrosivity
test
methods.
In
response,
the
ICCVAM
Dermal
Corrosion
Interagency
Working
Group
drafted
proposed
Performance
Standards
based
on
the
validated
test
methods
for
these
three
types
of
in
vitro
dermal
corrosivity
assays:
membrane
barrier
test
methods,
human
skin
model
system
test
methods,
and
skin
TER
test
methods
(
Refs.
10,
11,
12).
The
ICCVAM
Performance
Standards
include
the
essential
test
method
components
for
each
assay
and
the
subset
of
Reference
Chemicals
to
be
used
to
demonstrate
comparability
of
performance.
The
essential
test
method
components
can
be
used
as
a
basis
for
generic
guidelines.
ICCVAM
is
expected
to
finalize
the
Performance
Standards
for
these
corrosivity
assays
in
November
2003.
EPA
has
an
opportunity
to
provide
comments
on
these
draft
Performance
Standards
and
generic
guidelines
including
consideration
of
Scientific
Advisory
Panel
recommendations.
Page
7
of
13
OPPTS
will
revise
its
870.2500
test
guideline
for
Dermal
Irritation
to
incorporate
the
three
validated
in
vitro
methods
for
corrosivity,
drawing
upon
the
ICCVAM
Performance
Standards
for
the
three
assays.

New
test
methods
proposed
for
use
as
"
me­
too's"
based
on
each
of
these
corrosivity
assays
must
meet
the
Performance
Standards.
A
range
of
corrosive
substances
is
included
in
each
list
of
Reference
Chemicals.
Other
test
methods
meeting
the
Performance
Standards
should
have
reliability
and
performance
that
are
equivalent
to
or
better
than
that
of
each
validated
method.

III.
ENSURING
QUALITY
OF
IN
VITRO
ALTERNATIVE
TEST
METHODS
Special
Considerations
for
In
Vitro
Assays:

Testing
laboratories
must
use
good
scientific
practices,
namely
appropriate
calibration
and
standardization
methodology
from
a
variety
of
other
technical
disciplines
in
order
to
handle
the
elements
of
all
assay
systems.
For
example,
prior
to
running
an
unknown
chemical,
chemical
instrumentation
should
be
calibrated
in
accordance
with
the
manufacturer's
specifications
and
standard
samples
evaluated;
in
vitro
assay
using
cells
in
culture
should
use
good
cell
culture
practices;
etc.
Similarly,
performance
of
each
lot
of
an
in
vitro
system
should
also
be
"
calibrated"
to
assess
how
their
response
levels
can
be
used
to
predict
in
vivo
effects.

The
use
of
in
vitro
systems
to
replace
animal
tests
in
toxicology
testing
calls
for
meticulous
characterization
of
manufacturing
processes
or
isolation
and
handling
of
cells
in
culture,
tissue
constructs,
microchip
arrays,
and
the
like.
The
inherent
variation
of
in
vitro
test
systems
calls
for
special
standardization.
Target
tissues
for
in
vitro
systems
must
be
well­
characterized
and
identified,
maintained,
and
handled
under
appropriate
conditions
and
shown
to
be
viable
with
expected
responsiveness.

Cells
in
Culture
Cell
culture
test
systems
must
be
accompanied
by
provisions
to
assure
that
they
are
morphologically
and
physiologically
correct
and
that
they
show
the
responsiveness
to
chemicals
is
the
same
as
that
of
the
validated
cell
culture
system.
Assay
systems
that
use
normal
human
cells
(
e.
g.,
keratinocytes
or
hepatocytes)
have
special
challenges
in
that
these
cells
must
be
used
as
primary
or
early
passage
cultures.
This
often
precludes
comprehensive
cell
characterization,
making
the
user
laboratory
more
dependent
on
the
supplier
for
cell
characterization
and
safety
data
(
e.
g.,
evidence
of
freedom
from
pathogens).

Tissue
Constructs
Tissue
constructs,
such
as
EPISKIN
and
EpiDerm,
generally
have
a
very
short
shelf
life
and
are
produced
in
A
small@
batches
over
time.
These
factors
limit
the
number
of
replicate
tissues
that
each
user
can
dedicate
for
the
quality
control
efforts.
Thus,
the
user
is
dependent
upon
Page
8
of
13
the
manufacturer
to
supply
many
of
the
basic
elements
of
cell/
tissue
characterization
and
quality
control.
Such
data
should
include
information
on
cell
characterization,
tests
for
adventitious
agents,
structural
characteristics,
and
responses
to
positive
control
chemicals.
The
structural
and
functional
evaluation
should
be
performed
on
each
lot
of
tissue,
and
these
data
should
be
available
to
the
user
laboratory.
Even
with
these
procedures,
users
also
need
to
ensure
that
the
cells
or
tissues
are
functioning
within
normal
limits
after
shipping
and
handling
that
the
execution
of
the
assay
is
within
normal
limits.

Bioconstructs
Noncellular
bioconstructs
for
test
systems
are
generally
prepared
by
the
testing
laboratory.
It
is
critical
to
assure
that
morphology
and
responsiveness
is
within
acceptable
ranges
for
each
batch.
Manufacturers
of
kits
using
bioconstructs
should
recommend
tests
for
user
laboratories
so
that
performance
of
such
bioconstructs
can
be
assured.

Ex
Vivo
Tissues
Suppliers
of
ex
vivo
tissues
may
use
variable
sources
for
such
tissues
and
differences
in
handling
may
affect
tissue
viability.
Procedures
must
be
available
to
assure
viability
and
responsiveness
of
ex
vivo
systems
on
a
regular
basis.

Microassays
Currently
there
are
wide
variations
in
the
design,
data
extraction
and
analysis
for
microarrays
for
toxicogenomic
experiments.
This
calls
for
recording
of
sufficient
information
so
that
results
can
be
correctly
interpreted
or
replicated
(
Ref.
7).

Good
Laboratory
Practice
Regulations:

Quality
control
is
an
essential
element
for
any
regulated
study
so
that
results
of
the
assay
can
be
determined
to
be
meaningful
and
can
be
compared
with
data
from
previous
studies
within
laboratories
and
from
one
laboratory
to
another.
The
principles
of
GLP
have
been
agreed
internationally
to
promote
the
quality
and
relevance
of
test
data
used
for
determining
the
safety
of
chemicals
and
chemical
products
(
Ref.
13).
Agency
GLP
regulations
are
concerned
with
the
organizational
process
and
the
conditions
under
which
non­
clinical
health
and
environmental
safety
studies
are
planned,
performed,
recorded,
archived
and
reported
(
Ref.
4).
Their
aim
is
to
assure
consistency,
traceability
and
reproducibility
of
test
results.
Quality
control
procedures
for
studies
submitted
to
the
Agency
for
pesticides
and
industrial
chemicals
can
be
set
forth
in
OPPTS
test
guidelines,
and
included
in
Performance
Standards
established
by
ICCVAM
for
each
new
test
method
when
it
reviews
the
status
of
their
validations.
3
GLP
regulations
place
the
ultimate
responsibility
with
the
company
submitting
data
to
the
government
to
verify
that
the
assay
fulfills
regulatory
testing
needs.
GLP
and
good
science
responsibilities
of
the
testing
laboratories
(
users
of
in
vitro
systems)
include
using
a
predefined
study
protocol
for
the
assay,
training
the
technical
staff
and
ensuring
that
staff
skills
Page
9
of
13
are
maintained,
and
employing
concurrent
controls,
including
benchmark
controls,
if
appropriate,
to
monitor
assay
performance.

It
is
the
Agency's
assumption
that
the
manufacturer
of
the
assay
system
conducts
appropriate
quality
control
for
the
manufacturing
process
of
PTMs
in
order
to
ensure
that
test
systems
maintain
consistency
of
performance.
Data
for
each
batch
of
PTMs
demonstrating
test
system
performance
should
be
available
as
part
of
the
study
record.
If
not,
the
testing
laboratory
should
be
able
to
request
such
quality
control
documentation
from
the
manufacturer.
In
addition,
the
testing
laboratory
should
also
have
its
own
procedures
to
verify
performance
of
the
test
system
when
it
is
received
in
the
laboratory.

Use
of
Reference
Chemicals
to
Demonstrate
Test
Performance:

The
set
of
Reference
Chemicals
used
to
validate
a
particular
test
method
are
wellcharacterized
in
vivo
as
well
as
in
vitro
in
multiple
laboratories.
Such
Reference
Chemicals
are
expected
to
behave
in
a
consistent
manner
and
can
also
be
used
as
a
source
of
controls,
benchmark
chemicals,
and
training
or
calibration
sets.
(
See
definitions
below)

Test
guidelines
generally
call
for
use
of
positive
and
negative
controls.
When
available,
normative
ranges
of
historical
values
for
positive
controls
provide
Agency
reviewers
a
basis
for
assessing
the
way
the
test
system
performs
in
historical
context.
In
addition,
when
reviewing
results
for
test
chemicals,
Agency
reviewers
can
gain
extra
confidence
in
their
assessments
by
comparing
results
for
the
test
chemical
with
those
for
benchmark
chemicals
from
a
similar
chemical
class
but
with
various
potencies.
Therefore,
the
Agency
intends
to
require
use
of
concurrent
controls
in
its
guidelines
for
in
vitro
test
systems
and
also
recommends
use
of
benchmark
chemicals,
especially
for
assessment
of
test
chemicals
that
may
show
weak
or
negligible
responses.

Laboratories
using
new
in
vitro
methods
(
proprietary
or
otherwise)
can
demonstrate
that
the
test
is
being
performed
properly
by
using
training
or
calibration
sets
and
comparing
results
with
those
found
during
validation
trials.

Controls:

Controls
may
be
positive
or
negative.
For
in
vitro
studies,
positive
control
response(
s)
are
part
of
the
process
of
demonstrating
the
functional
integrity
of
target
tissues,
proper
treatment
of
the
cells
or
tissue,
and
proper
execution
of
the
test
method.
Negative
control
responses
are
often
used
to
set
the
baseline
of
cell
or
tissue
response
against
which
the
responses
of
the
cells
or
tissues
treated
with
the
test
article
or
positive
control
can
be
compared.

One
would
expect
that
controls
address
the
endpoint
reported
in
the
assay
and
can
be
used
to
provide
a
measure
of
the
performance
of
the
assay
at
each
run.
Concurrent
positive
and
negative
controls
should
be
used
for
each
trial
using
the
assay.
In
that
way,
they
help
to
establish
whether
a
valid
trial
was
performed
when
test
data
for
the
unknown
chemical
are
Page
10
of
13
submitted
to
regulatory
agencies.
In
addition,
results
of
control
trials
can
be
compared
with
historical
data
and
used
for
trend
analysis
so
that
any
drift
in
the
assay
system
can
be
detected.

The
positive
control
should
be
able
to
detect
over­
and
under­
response.
A
trial
where
the
control
values
falls
outside
the
acceptable
limits
would
be
repeated
and
the
data
from
such
a
trial
would
not
be
included
in
a
regulatory
submission.
This
avoids
the
inclusion
of
spurious
data
from
a
trial
that
is
outside
the
normal
limits
of
the
assay.
In
some
types
of
assays
(
e.
g.,
cytotoxicity
studies),
the
negative
control
is
used
to
normalize
the
measure
of
cell
viability
(
e.
g.,
dye
uptake)
and
so
the
acceptance
criteria
focus
on
the
performance
of
the
positive
control.

Benchmark
Class
and
Potency
Chemicals:

Benchmark
materials
are
not
a
substitute
for
positive
controls,
but
are
a
valuable
addition
to
an
assay.
Whereas
controls
provide
a
measure
of
stability
of
the
in
vitro
assay
system,
the
appropriate
benchmark
chemicals
demonstrate
a
range
of
acceptable
responses
for
each
class
of
chemicals
for
which
the
assay
is
valid.
Benchmark
chemicals
should
be
selected
from
chemicals
that
are
well
characterized
for
the
assay,
i.
e.,
the
Reference
Chemicals.
They
differ
from
positive
controls
in
that
they
are
matched
to
the
chemical
class
of
the
test
material
in
each
test
trial
and
are
used
to
set
upper
and/
or
lower
limits
of
response
against
which
the
response
of
the
unknown
chemical
may
be
judged.
The
upper
limit
(
and
lower
limit
if
applicable)
of
each
benchmark
is
set
relative
to
acceptable
responses
in
vivo.

Calibration
Set:

Calibration
of
elements
of
a
test
system
or
method
may
be
warranted
for
certain
in
vitro
test
methods.
For
example,
the
apparatus
used
to
probe
or
measure
the
endpoint
or
to
augment
an
ex
vivo
tissue
or
tissue
construct
may
not
be
standardized.
In
such
cases,
such
detection
or
support
equipment
can
be
calibrated
using
a
calibration
set,
which
is
a
suitable
subset
of
the
Reference
Chemicals
used
to
validate
the
test.

Training
Set:

When
laboratories
begin
to
use
a
validated
test
method
or
test
kit,
a
training
set
drawn
from
the
list
of
Reference
Chemicals
for
the
method
can
be
used
to
learn
the
method
or
refine
testing
techniques.
Once
use
of
the
new
method
is
established
in
the
laboratory
and
instrumentation
and
procedures
calibrated,
use
of
controls
and
benchmarks
with
each
test
trial
should
be
sufficient.

Consideration
of
Quality
Issues
by
ICCVAM,
ECVAM,
and
OECD:

Organizations
in
the
United
States
and
Europe
have
been
evaluating
approaches
to
ensure
integrity
and
performance
of
new
in
vitro
methods
when
they
are
proposed
to
fulfill
regulatory
test
requirements
as
alternatives
to
animal
testing.
These
actions
can
be
expected
to
help
the
testing
laboratories
(
i.
e.,
study
directors,
technical
staff)
in
developing
processes
to
ensure
Page
11
of
13
compliance
with
GLP
principles
and
provide
regulatory
scientists
with
specific
information
and
guidance
to
better
assess
the
quality
of
in
vitro
studies
and
the
authenticity
of
in
vitro
study
results
submitted
to
them
for
regulatory
purposes.

In
the
course
of
executing
its
mission
to
develop
and
promote
in
vitro
studies
as
alternatives
for
conventional
animal
testing,
the
ECVAM)
has
been
heavily
involved
in
considering
quality
control
measures
specifically
for
in
vitro
studies.
In
response
to
a
recommendation
by
ECVAM
in
coordination
with
ICCVAM,
the
Organization
for
Economic
Cooperation
and
Development
(
OECD)
will
develop
a
consensus
document
to
interpret
the
principles
of
GLP
for
execution
of
in
vitro
assays
(
Ref.
14).
The
report
of
the
ECVAM
workshop
(
Ref.
15)
on
principles
of
GLP
when
applied
to
in
vitro
toxicology
provides
several
examples
of
areas
where
specific
guidance
for
in
vitro
studies
might
be
incorporated
into
the
new
OECD
consensus
document
for
GLP.

ECVAM
has
also
issued
a
report
on
Good
Cell
Culture
Practice
(
GCCP)
(
Ref.
6).
The
GCCP
report
specifies
procedures
to
ensure
that
test
systems
are
free
of
any
contamination
or
other
diseases
or
conditions
at
the
beginning
of
the
study
that
might
interfere
with
the
outcome
of
the
study
and
calls
for
the
origin
(
species/
tissue),
source,
arrival
condition
and
maintenance
requirements
to
be
documented
and
confirmed
at
the
laboratory
on
a
regular
basis.
Documentation
of
critical
cell
culture
parameters
is
expected
to
help
regulatory
authorities
in
the
acceptance
and
interpretation
of
in
vitro
data.
In
addition,
ECVAM
in
cooperation
with
ICCVAM
is
planning
to
convene
a
series
of
workshops
to
develop
special
technical
guidance
for
in
vitro
studies.

References.

1.
Interagency
Coordinating
Committee
for
Validation
of
Alternative
Methods.
1997.
Validation
and
Regulatory
Acceptance
of
Toxicological
Methods
of
Test
Methods.
NIH
Publication
97­
3981.
See
Chapter
2.
Validation
of
Test
Methods
and
Chapter
3.
Regulatory
Acceptance
of
Toxicological
Test
Methods.

2.
Interagency
Coordinating
Committee
for
Validation
of
Alternative
Methods.
2003.
ICCVAM
Guidelines
for
the
Nomination
and
Submission
of
New,
Revised
and
Alternative
Test
Methods.
NIH
Publication
03­
4508.
September
2003.

3.
Environmental
Protection
Agency.
Ethics
Advisory
95­
18,
Use
of
Public
Office
for
Private
Gain
­
Endorsements.
Based
on
provisions
of
the
"
Standards
of
Ethical
Conduct
for
Employees
of
the
Executive
Branch"
at
5
CFR
2635.702
and
on
provisions
of
5
CFR
2635.703.

4.
Environmental
Protection
Agency.
40
CFR
160
Good
Laboratory
Practice
Standards
(
Office
of
Pesticides)
and
40
CFR
792
Good
Laboratory
Practice
Standards
(
Office
of
Prevention
and
Toxic
Substances).

5.
Environmental
Protection
Agency.
Order
5360.1
A2.
Policy
and
Program
Page
12
of
13
Requirements
for
the
Mandatory
Agency­
Wide
Quality
System.
May
2000.

6.
Hartung,
T.,
Balls,
M.,
Bardouille,
C.,
Blanck,
O.,
Coecke,
S.,
Gstraunthaler,
G.,
and
Lewis,
D.
(
2003).
Good
Cell
Culture
Practice:
ECVAM
Good
Cell
Culture
Practice
Task
Force
Report
1.
ATLA
30:
407­
414.

7.
Minimum
Information
About
a
Microarray
Experiment
­
MIAME
for
Toxicogenomics(
MIAME/
TOX).
Draft
report
February
12,
2003.
Available
at
http://
www.
mged.
org/.

8.
Interagency
Coordinating
Committee
for
Validation
of
Alternative
Methods.
2002.
ICCVAM
Evaluation
of
EPISKINTM,
EpiDermTM
(
EPI­
200)
,
and
the
Rat
Skin
Transcutaneous
Electrical
Resistance
(
TER)
Assay:
In
Vitro
Test
Methods
for
Assessing
Dermal
Corrosivity
Potential
of
Chemicals.
NIH
Publication
No.
02­
4502.
June
2002.
Available
at
http://
iccvam.
niehs.
nih.
gov/
methods/
epiddocs/
cwgfinal/
cwgfinal.
pdf
9.
Interagency
Coordinating
Committee
for
Validation
of
Alternative
Methods.
1999.
Corrositex

:
An
In
Vitro
Test
Method
for
Assessing
Dermal
Corrosivity
Potential
of
Chemicals.
NIH
Publication
No.
99­
4495.
June
1999.
Available
at:
http://
iccvam.
niehs.
nih.
gov/
docs/
reports/
corprrep.
pdf.

10.
Interagency
Coordinating
Committee
for
Validation
of
Alternative
Methods.
2003.
ICCVAM
Minimum
Performance
Standards:
In
Vitro
Membrane
Barrier
Test
Systems
for
Skin
Corrosion.

11.
Interagency
Coordinating
Committee
for
Validation
of
Alternative
Methods.
2003.
ICCVAM
Minimum
Performance
Standards:
In
Vitro
Human
Skin
Model
Systems
for
Skin
Corrosion.

12.
Interagency
Coordinating
Committee
for
Validation
of
Alternative
Methods.
2003.
ICCVAM
Minimum
Performance
Standards:
In
Vitro
Skin
Transcutaneous
Electrical
Resistance
(
TER)
Tests
for
Skin
Corrosion.

13.
Organization
for
Economic
Cooperation
and
Development.
1981.
Decision
of
the
Council
Concerning
the
Mutual
Acceptance
of
Data
in
the
Assessment
of
Chemicals,
adopted
May
12,
1981.

14.
Interagency
Coordinating
Committee
for
Validation
of
Alternative
Methods.
Memorandum
to
OECD
dated
February
19,
2003.
Justification
for
Development
of
An
OECD
Guidance
Document
on
the
Application
of
Good
Laboratory
Practice
(
GLP)
Principles
to
In
Vitro
Testing.

15.
Cooper­
Hannan,
R.,
Harbell,
J.
W.,
Coecke,
S.,
Balls,
M.,
Bowe,
G.,
Cervinka,
M.,
Clothier,
R.,
Hermann,
F.,
Klahn,
L.
K.,
de
Lange,
J.,
Lievsch,
M
and
Page
13
of
13
Vanparys,
P.
(
1999).
The
principles
of
Good
Laboratory
Practice:
Application
to
in
vitro
toxicology
studies.
The
report
and
recommendations
of
ECVAM
workshop
37.
ATLA
27,
539­
577.

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