ArticlePDF Available
Meetinci
Report
The
Three
Rs:
The
Way
Forward
Joanne
Zurlo,
Deborah
Rudacille,
and
Alan
M.
Goldberg
Center
for
Alternatives
to
Animal
Testing,
Department
of
Environmental
Health
Sciences,
Johns
Hopkins
School
of
Public
Health,
Baltimore,
MD
21202-6709
USA
Laws
that
mandate
replacement
alterna-
tives,
reduction
alternatives,
and
refinement
alternatives
(the
Three
Rs)
in
scientific
research
have
been
passed
in
the
United
Kingdom,
Germany,
the
Netherlands,
the
United
States,
and
the
European
Union
over
the
past
decade.
Full
implementation
of
this
newly
developed
legislation
depends
upon
scientists'
ability
to
understand
ani-
mal
welfare
issues
and
to
accept
the
legiti-
macy
of
the
public's
interest
in
the
conduct
of
science.
The
European
Centre
for
the
Validation
of
Alternative
Methods
(ECVAM),
established
by
the
European
Commission
in
1991
to
promote
the
scien-
tific
and
regulatory
acceptance
of
alternative
methods,
recently
sponsored
a
workshop
to
discuss
the
current
status
of
the
Three
Rs
and
to
make
recommendations
aimed
at
achieving
greater
acceptance
of
the
concept
of
humane
experimental
technique.
Twenty-one
scientists
professionally
committed
to
the
Three
Rs
were
invited
to
attend
the
conference,
which
was
chaired
by
Michael
Balls,
head
of
ECVAM,
and
Alan
M.
Goldberg,
director
of
the
Johns
Hopkins
Center
for
Alternatives
to
Animal
Testing
(CAAT).
The
conference
was
held
in
Sheringham,
Norfolk,
UK
on
May
30,
1995-June
3,
1995.
A
report
based
on
the
conference
was
published
by
ECVAM
(1)
in
December
1995
and
is
excerpted
as
follows.
Origins
of
the
Three
Rs
The
Three
Rs
originated
in
a
proposal
made
in
1954
by
Charles
Hume,
founder
of
the
Universities
Federation
for
Animal
Welfare
(UFAW),
that
the
UFAW
should
undertake
a
scientific
study
of
humane
technique
in
laboratory
animal
experiments.
The
project
was
managed
by
a
committee
under
the
chairmanship
of
Sir
Peter
Medawar,
the
Nobel
prize
winning
immunologist,
with
William
Lane-Petter,
Secretary
of
the
Research
Defence
Society
of
Great
Britain,
among
its
members.
Christine
Stevens,
founder
of
the
Animal
Welfare
Institute
(AWI)
in
the
U.S.,
provided
financial
sup-
port
for
the
project.
W.M.S.
Russell,
a
zool-
ogist,
and
R.
L.
Burch,
a
microbiologist,
were
appointed
to
carry
out
the
work,
which
led
to
the
publication
of
the
book
The
Principles
of
Humane
Experimental
Technique
(2)
in
1959.
At
the
time
of
the
book's
publication,
Charles
Hume
commented
that
This
deserves
to
become
a
classic
for
all
time,
and
we
have
great
hopes
that
it
will
inaugurate
a
new
field
of
systematic
study.
We
hope
that
others
will
follow
up
the
lead
it
has
given,
and
that
a
generalised
study
of
humane
technique,
as
a
systematic
compo-
nent
of
the
methodology
of
research,
will
come
to
be
considered
essential
to
the
training
of
a
biologist.
Hume's
predictions
regarding
the
book's
impact
have
been
realized
as
the
concepts
of
replacement
alternatives,
reduction
alternatives,
and
refinement
alternatives
have
become
established
in
law.
However,
at
the
present
time,
a
thorough
working
knowledge
and
acceptance
of
the
principles
of
humane
experimental
technique
among
scientists
in
general
remains
at
best
elusive
and
at
worst
ignored.
Scientific
and
Ethical
Justification
Current
legislation
in
Europe
and
the
United
States
decrees
that
all
proposed
use
of
laboratory
animals
should be
subject
to
review
to
determine
whether
such
use
appears
to
be
scientifically
and
ethically
justifiable.
Individually
and
collectively,
such
laws
not
only
recognize
Russell
and
Burch's
concept
(2)
but
place
legal
and
moral
obligations
on
all
concerned
to
replace,
reduce,
and
refine
laboratory
ani-
mal
experimentation
wherever
possible.
The
degree
to
which
proposed
animal
use
is
reviewed
varies
from
country
to
country.
For
example,
in
the
United
Kingdom,
a
working
party
of
the
Institute
of
Medical
Ethics
concluded
that
a
project
using
animal
subjects
should
only
be
done
when
the
review
committee
ascertains
that
the
aim
of
the
project
is
worthwhile;
that
the
experimental
design
of
the
project
is
such
that
there
is
a
good
likelihood
of
achieving
the
stated
aims;
that
the
aim
could
not
be
achieved
using
methods
or
subjects
that
were
morally
more
acceptable
and
that
produced
no
less
scientifically
acceptable
results;
and
that
the
likely
bene-
fits
of
the
project
are
worth
the
costs
to
the
animals
in
terms
of
pain
and
suffering
(3).
In
the
United
States,
the
Animal
Welfare
Act
requires
that
all
procedures
involving
animals
be
reviewed
by
an
insti-
tutional
animal
care
and
use
committee
(IACUC).
The
IACUCs
are
required
to
ensure
that
the
protocols
are
worthwhile,
that
they
use
the
minimum
number
of
ani-
mals
necessary,
and
that
the
investigators
document
that
they
have
adequately
consid-
ered
alternatives
to
any
procedure
that
caus-
es
more
than
momentary
pain
or
distress
(either
with
or
without
the
use
of
anesthet-
ics).
Guidelines
for
searching
for
alternative
procedures
have
been
prepared
to
assist
investigators
and
IACUC
members
in
these
considerations
(4).
The
Three
Rs
Reduction
Alternatives
The
term
reduction
alternatives
describes
methods
for
obtaining
comparable
levels
of
information
from
the
use
of
fewer
animals
in
scientific
procedures
or
for
obtaining
more
information
from
a
given
number
of
animals
so
that,
in
the
long
run,
fewer
ani-
mals
are
needed
to
complete
a
given
research
project
or
test.
The
greater
the
number
of
animals
used,
the
greater
will
be
the
overall
costs
in
terms
of
animal
suffer-
ing.
Therefore,
the
number
of
animals
used
should be
the
minimum
that
is
consistent
with
the
aims
of
the
experiment.
There
is
evidence
that
poor
experimental
design
and
inappropriate
statistical
analysis
of
experimental
results
leads
to
inefficient
use
of
animals
and
scientific
resources
in
toxico-
logical
research
(5,6).
Previous
studies
of
sta-
tistical
methods
used
in
other
areas
of
bio-
medical
research
reveal
similar
findings
(7).
In
some
cases,
the
level
of
statistical
expertise
appears
to
be
so
low
that
investigators
are
either
unaware
of
the
potential
value
of
obtaining
statistical
advice,
or
they
are
unable
to
obtain
appropriate
statistical
advice
because
there
are
so
few
biometricians
with
experience
in
their
field
of
interest.
A
basic
understanding
of
experimental
design
and
statistics
is
necessary
for
all
scien-
tists.
For
investigators
with
no
previous
training
in
statistics,
this level
of
expertise
can
probably
be
obtained
from
an
introduc-
tory
course.
There
are
many
texts
on
statisti-
cal
methods,
which
can
be
used
for
both
learning
purposes
and
as
reference
books.
Biomedical
research
workers
should
have
more
detailed
training
in
biometrics
and
sta-
Address
correspondence
to
J.
Zurlo,
Center
for
Alternatives
to
Animal
Testing,
Department
of
Environmental
Health
Sciences,
Johns
Hopkins
School
of
Public
Health,
111
Market
Place,
Suite
840,
Baltimore,
MD
21202-6709
USA.
Volume
104,
Number
8,
August
1996
*
Environmental
Health
Perspectives
878
Meeting
Report.
The
Three
Rs
tistics
so
that
they
can
act
as
consultants
to
other
investigators
in
their
own
institutes.
Refinement
Alternatives
Refinement
alternatives
encompass
those
methods
that
alleviate
or
minimize
poten-
tial
pain
and
distress
and
enhance
animal
well-being.
Distress
is
an
aversive
state
in
which
an
animal
is
unable
to
adapt
com-
pletely
to
stressors
and
the
resulting
stress
and,
therefore,
shows
maladaptive
behav-
ior.
The
stressors
may
induce
physiological,
psychological,
or
environmental
stress.
Pain
results
from
potential
or
actual
tissue
dam-
age,
such
as
that
caused
by
injury,
surgery,
or
disease,
and
can
lead
to
distress
(8-10).
Much
potential
pain
and
distress
can
be
avoided
or
alleviated
with
the
proper
use
of
anesthetics,
analgesics,
and
tranquilizers.
This
critical
component
of
any
comprehen-
sive
program
of
veterinary
care
provides
for
frequent
observation
of
the
animals
by
trained
veterinary
staff
to
detect
and
relieve
pain
and
distress.
However,
a
substantial
number
of
animals
used
in
research
and
test-
ing
experience
unrelieved
pain
and
distress.
At
present,
we
do
not
have
a
conve-
nient
and
standardized
way
of
objectively
assessing
animal
pain
and
distress.
Rather,
the
assessment
is
generally
based
on
subjec-
tive
clinical
signs
of
abnormal
behavior
and
appearance.
Although
the
implementation
of
refinement
alternatives
depends
largely
on
the
ability
of
scientists
to
observe
and
understand
the
behavior
and
needs
of
labo-
ratory
animals,
many
experimenters
are
as
lacking
in
ethological
knowledge
as
they
are
in
statistical
training.
The
best
approach
to
pain
and
distress
is
to
assume
that
a
procedure
that
inflicts
pain
and
dis-
tress
in
human
beings
will
inflict
at
least
as
much
pain
and
distress
in
animals,
unless
there
is
evidence
to
the
contrary.
Very
little
research
finding
is
available
to
support
efforts
to
investigate
and
refine
experimental
techniques
and
scientific
proce-
dures.
Furthermore,
there
is
no
readily
avail-
able
up-to-date
knowledge
base
on
refine-
ment.
Techniques
that
are
developed
to
refine
a
procedure
are
frequently
not
reported
in
the
scientific
literature
or
are
established
simply
as
standard
operating
procedures
(SOPs)
within
an
institution.
To
establish
best
practice
and
to
advance
the
implementa-
tion
of
refinement
altematives,
it
is
important
to
share
such
experience,
data,
and
SOPs.
Sharing
of
data
and
theories
is
normally
accomplished
via
the
scientific
literature,
but
there
has
been
a
marked
lack
of
opportunity
to
discuss
and
provide
information
on
refine-
ment
alternatives
in
the
main
biological
jour-
nals.
Consequently,
scientists
are
not
suffi-
ciently
aware
of
the
concept
of
refinement
alternatives
and,
in
general,
they
do
not
rec-
ognize
the
importance
of
refinement
in
their
research.
The
concept
of
recognizing,
mini-
mizing,
and
eliminating
pain
and
distress
in
laboratory
animals
should
be
included
in
training
programs
for
all
persons
involved
in
the
care
and
use
of
laboratory
animals.
Details
of
refinement
and
animal
welfare
considerations
should
routinely
be
induded
in
scientific
papers
and
publications.
Replacement
Alternatives
Replacement
alternatives
encompass
those
methods
that
permit
a
given
purpose
to
be
achieved
without
conducting
experiments
or
other
scientific
procedures
on
animals.
Russell
and
Burch
(2)
distinguished
between
relative
replacement,
e.g.,
the
humane
killing
of
a
vertebrate
animal
to
provide
cells,
tissues,
or
organs
for
in
vitro
studies
and
absolute
replacement
in
which
animals
would
not
need
to
be
used
at
all,
e.g.,
the
culture
of
human
and
invertebrate
cells
and
tissues.
The
range
of
replacement
alternative
methods
and
approaches
includes
the
improved
storage,
exchange,
and
use
of
information
about
previous
animal
experi-
ments
to
avoid
unnecessary
repetition
of
animal
procedures;
use
of
physical
and
chemical
techniques
and
predictions
based
upon
the
physical
and
chemical
properties
of
molecules;
use
of
mathematical
and
computer
models;
use
of
organisms
with
limited
sentience
such
as
invertebrates,
plants
and
microorganisms;
use
of
in
vitro
methods
including
subcellular
fractions,
tissue
slices,
cell
suspensions,
and
perfused
organs;
and
human
studies
including
use
of
human
volunteers,
postmarketing
surveil-
lance,
and
epidemiology.
In
many
areas
of
the
biomedical
sci-
ences,
in
vitro
methods
are
increasingly
used
as
the
methods
of
choice
in
place
of
animal
studies.,
not
because
they
provide
precisely
the
same
information,
but
because
they
offer
the
best
scientific
approach.
Russell
and
Burch
(2)
discussed
the
relative
merits
of
fidelity
and
discrimination
mod-
els,
noting
that
high-fidelity
models,
as
exemplified
by
the
use
of
rodents
and
other
laboratory
mammals
in
toxicity
testing,
are
used
because,
in
their
general
physiological
and
pharmacological
properties,
they
are
similar
to
humans.
High
discrimination
models,
on
the
other
hand,
"reproduce
one
particular
property
of
the
original,
in
which
we
happen
to
be
interested"
(2).
Russell
and
Burch
(2)
warned
of
the
high-fidelity
fallacy
and
of
the
danger
of
expecting
discrimination
in
particular
cir-
cumstances
from
models
that
show
high
fidelity
in
other,
more
general
terms-a
prediction
illustrated
by
recent
analyses
of
the
differing
molecular
responses
to
certain
chemicals
by
the
rat,
the
mouse,
and
the
human.
Russell
and
Burch
(2)
pointed
out
that
the
fidelity
of
mammals
as
models
for
man
is
greatly
overestimated;
however,
replacement
alternative
methods
must
be
based
on
good
science,
and
extravagant
claims
that
cannot
be
substantiated
must
be
avoided.
The
development
and
acceptance
of
replacement
alternatives
for
both
research
and
testing
must
be
based
on
a
suf-
ficient
understanding
of
the
molecular
and
cellular
mechanistic
basis
of
what
is
being
studied
or
measured,
i.e.,
on
sound
science.
Education
and
Training
The
successful
implementation
of
the
Three
Rs
depends
upon
the
education
and
train-
ing
of
those
involved
in
research
and
test-
ing.
Education
is
defined
as
the
didactic
presentation
of
the
information
and
theo-
ries
of
animal
use
that
will
contribute
to
the
development
of
proper
attitudes
toward
the
use
of
animals
in
scientific
procedures.
Training
is
defined
as
the
acquisition
of
practical
knowledge
and
skill
directly
associ-
ated
with
animal
handling
and
procedures.
The
objective
of
the
education
and
training
is
to
provide
sufficient
information
to
allow
scientists
to
conduct
animal
proce-
dures
to
high
standards
of both
science
and
animal
welfare,
following
proper
evaluation
of
the
scientific
and
ethical
considerations
that
should
govern
the
use
of
laboratory
animals.
Coursework
should
contribute
to
a
scientist's
ability
to
design
experiments
properly
and
to
plan
research
strategies,
to
become
competent
in
animal
handling
and
the
performance
of
scientific
procedures,
to
make
decisions
with
regard
to
the
ethics
of
using
animals
in
experiments,
and
to
deter-
mine
whether
alternatives
are
available.
A
description
of
the
course
on
animal
experimentation
and
alternatives
currently
offered
at
Utrecht
University
in
The
Netherlands
(11)
and
the
guidelines
of
the
Federation
of
European
Laboratory
Animal
Science
Associations
(FELASA)
(12)
and
the
U.S.
National Research
Council
(13)
could
serve
as
prototypes
for
the
develop-
ment
of
courses
in
other
countries.
The
Way
Forward
The
use
of
the
term
alternatives
to
encom-
pass
all
of
the
Three
Rs
is
now
widely
accept-
ed
in
many
countries,
enshrined
in
legisla-
tion,
and
incorporated
into
the
names
of
var-
ious
centers
throughout
the
world.
However,
some
scientists
see
its
use
as
being
driven
by
political
and
social
forces
rather
than
by
sci-
entific
issues.
This
is
partly
due
to
a
lack
of
appreciation
of
the
basis
of
the
Three
Rs
concept
as
proposed
by
Russell
and
Burch
(i.e.,
that
scientific
excellence
and
the
greatest
humanity
in
the
use
of
laboratory
animals
are
inextricably
linked)
(2).
It
also
stems
Environmental
Health
Perspectives
*
Volume
104,
Number
8,
August
1996
879
Meeting
Report
*
Zurlo
et
al.
from
a
defensive
attitude
among
some
scien-
tists,
perhaps
resulting
from
the
campaigns
of
some
antivivisection
organizations
and
from
insufficient
dialogue
among
the
scien-
tific
and
animal
protection
communities.
In
the
mid-1990s,
the
question
we
face
is
whether
there
will
be
a
revolution
in
thinking
and
practice,
which
is
what
is
needed
if
the
principles
of
humane
experi-
mental
technique
are
to
be
brought
fully
and
effectively
into
operation.
Much
has
been
achieved,
but
there
is
still
consider-
able
room
for
progress
and
improvement.
The
Sheringham
workshop
participants
propose
several
general
recommendations:
*
Existing
laboratory
animal
protection
laws
should
be
fully
implemented.
*
All
countries
should
have
a
legal
frame-
work
that
actively
incorporates
the
Three
Rs
into
all
animal-based
research,
testing,
and
education.
*
There
should
be
formal
and
informal
mechanisms
for
the
education
and
train-
ing
of
academic,
industrial,
and
govern-
ment
scientists
and
officials
in
the
Three
Rs
to
ensure
compliance
with
the
spirit
and
letter
of
laboratory
animal
protec-
tion
legislation
and
regulations.
*
There
should
be
international
discussion
and
agreement
on
what
levels
of
animal
suffering
should
not
be
permitted
in
any
circumstances,
regardless
of
any
likely
or
potential
benefits.
*
It
is
unacceptable
to
export
scientific
work
involving
laboratory
animals
to
avoid
scientifically
realistic,
but
more
stringent,
animal
welfare
codes.
The
participants
of
the
Sheringham
workshop
unanimously
reaffirmed
the
prin-
ciples
put
forth
by
Russell
and
Burch
(2)
that
humane
science
is
good
science
and
that
this
is
best
achieved
by
vigorous
appli-
cation
of
the
Three
Rs.
The
only
acceptable
animal
experiment
is
one
that
uses
the
smallest
number
of
animals
and
causes
the
least
possible
pain
or
distress,
is
consistent
with
the
achievement
of
a
justifiable
scientif-
ic
purpose,
and
is
necessary
because
there
is
no
other
way
of
achieving
that
purpose.
Any
proposed
experiment
on
animals
should
be
subjected
to
prior
and
effective
expert
review
by
an
ethics
committee.
Scientists
should
be
better
informed
about
the
Three
Rs
concept
and
should
be
encouraged
to
see
it
as
an
opportunity
for
reaping
benefits
of
every
kind-scientific,
economic,
and
humanitari-
an.
Only
in
this
way
can
the
aspirations
of
all
those
who
have
worked
for
the
good
of
both
human
and
animal
welfare
be
achieved
at
last.
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t1PIL
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#'S''"s"''s'#'$
,S
r
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~~~...
.............. ..........
...s
#sit,,.
t¢,,,1g$
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X
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tsk
s¢as
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$¢ssg:~~~~~~.
......
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:zX
3p;
.......
s
St-w, ,
ta~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.sS-
S$
............iX$S
-i,g,- ..
.
U..,...
~~~~~~~~~~~~~~~~~~~~~~~~~~~....
~ ~
~
~
~
.....
S$'S
'S.$||^s,$|.,,$¢
S,
41..,,,
,,,l
k
$'S
i
SSS
S
.....................
S,
S,
ffiSiS
S##
........................................,.;.....$
s~~...................
,.S~~~
$,
1Sj'.S
S;
,S
,
=
M
K
g
S.2
t$w'
'"$"t'
SS'-".l'|S#",.l
Xg.S$g
#£,IS
,]
$,,
[18"$
s :.
;sg
Zs.
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August
1996
*
Environmental
Health
Perspectives
... Efficient models are needed during the pre-clinical drug development process to evaluate basic drug-like characteristics, such as membrane permeation properties. There is growing compliance among the animal science community with the "Three Rs" principle, which promotes replacing animals with alternative models such as in vitro cell cultures (Dolberg & Reichl, 2016;Zurlo, Rudacille, & Goldberg, 1996). ...
Nasal drug administration has been identified as a potential alternative to oral drug administration, especially for systemic delivery of large molecular weight compounds. Major advantages of nasal drug delivery include high vascularity and permeability of the epithelial membranes as well as circumvention of first-pass metabolism. RPMI 2650 cell layers (in vitro cell model) and excised sheep nasal mucosal tissues (ex vivo sheep model) were evaluated with regard to epithelial thickness, selected tight junction protein expression (i.e. claudin-1, F-actin chains, zonula occludin-1), extent of p-glycoprotein (P-gp) related efflux of a model compound (Rhodamine-123, R123) and paracellular permeation of a large molecular weight model compound (FITC-dextran 4400, FD4). The cell model grown under liquid cover conditions (LCC) was thinner (24 ± 4 μm) than the epithelial layer of the sheep model (53 ± 4 μm), whereas the thickness of cell model grown under air liquid interface (ALI) conditions (53 ± 8 μm) compared well with that of the sheep model. Although the location and distribution of tight junction proteins and F-actin differed to some extent between the cell model grown under ALI conditions and the sheep model, the extent of paracellular permeation of FD4 was similar (Papp = 0.48 × 10⁻⁶ cm.s⁻¹ and 0.46 × 10⁻⁶ cm.s⁻¹, respectively). Furthermore, the bi-directional permeation of R123 yielded the same efflux ratio (ER = 2.33) in both models. The permeation results from this exploratory study indicated similarity in terms of compound permeation between the RPMI 2650 nasal epithelial cell line and the excised sheep nasal epithelial tissue model.
... While not critically sized, this scenario enabled the assessment of scaffold osseointegration and neotissue infiltration along the defect perimeter. This also reduced the number of animals utilized as two bilateral defects could be produced, rather than one central, critical-size defect (d = 15 mm), thus demonstrating a commitment to the 3R's principle of humane animal research [42,43]. Twelve rabbits were divided into three treatment groups (4 per group, N = 8 defects per group) with the prior group completed before beginning the next treatment group. ...
Article
Self-fitting scaffolds prepared from biodegradable poly(ε-caprolactone)-diacrylate (PCL-DA) have been developed for the treatment of craniomaxillofacial (CMF) bone defects. As a thermoresponsive shape memory polymer (SMP), with the mere exposure to warm saline, these porous scaffolds achieve a conformal fit in defects. This behavior was expected to be advantageous to osseointegration and thus bone healing. Herein, for an initial assessment of their regenerative potential, a pilot in vivo study was performed using a rabbit calvarial defect model. Exogenous growth factors and cells were excluded from the scaffolds. Key scaffold material properties were confirmed to be maintained following gamma sterilization. To assess scaffold integration and neotissue infiltration along the defect perimeter, non-critically sized (d = 8 mm) bilateral calvarial defects were created in 12 New Zealand white rabbits. Bone formation was assessed at 4 and 16 weeks using histological analysis and micro-CT, comparing defects treated with an SMP scaffold (d = 9 mm x t = 1 or 2 mm) to untreated defects (i.e. defects able to heal without intervention). To further assess osseointegration, push-out tests were performed at 16 weeks and compared to defects treated with poly(ether ether ketone) (PEEK) discs (d = 8.5 mm x t = 2 mm). The results of this study confirmed that the SMP scaffolds were biocompatible and highly conducive to bone formation and ingrowth at the perimeter. Ultimately, this resulted in similar bone volume and surface area versus untreated defects and superior performance in push-out testing versus defects treated with PEEK discs. Statement of significance Current treatments of craniomaxillofacial (CMF) bone defects include biologic and synthetic grafts but they are limited in their ability to form good contact with adjacent tissue. A regenerative engineering approach using a biologic-free scaffold able to achieve conformal fitting represents a potential “off-the-shelf” surgical product to heal CMF bone defects. Having not yet been evaluated in vivo, this study provided the preliminary assessment of the bone healing potential of self-fitting PCL scaffolds using a rabbit calvarial defect model. The study was designed to assess scaffold biocompatibility as well as bone formation and ingrowth using histology, micro-CT, and biomechanical push-out tests. The favorable results provide a basis to pursue establishing self-fitting scaffolds as a treatment option for CMF defects.
... Additionally, this retrospective study design supports animal reduction in research, which supports the United States legislative mandate to incorporate the three Rs (reduction, refinement, and replacement) into research [30]. Since study design limited the available specimens for BHS to archived FFPE tissue, the opportunistically collected DS lung tissues were similarly fixed and processed. ...
Article
Full-text available
Mycoplasma ovipneumoniae is a respiratory pathogen that impacts domestic sheep (Ovis aries; DS) and bighorn sheep (Ovis canadensis; BHS). BHS are reported to be more susceptible than DS to developing polymicrobial pneumonia associated with M. ovipneumoniae infection. Using formalin-fixed paraffin-embedded tissues, we performed a retrospective study investigating the pulmonary immune response of DS and BHS to M. ovipneumoniae infection. M. ovipneumoniae infected DS exhibited a more robust and well-organized BALT formation as compared to BHS. Digital analysis of immunohistochemical chromogen deposition in lung tissue was used to quantitate T cell marker CD3, B cell markers CD20 and CD79a, macrophage markers CD163 and Iba1, and cytokine IL-17. A significant interaction of species and infection status was identified for CD3, CD163, and IL-17. BHS had a greater increase in bronchiolar CD3 and bronchiolar and alveolar CD163 with infection, as compared to DS. BHS had an increase in bronchiolar associated lymph tissue (BALT) and alveolar IL-17 with infection, while these remained similar in DS regardless of infection status. IL-17 in respiratory epithelium of bronchi and bronchioles comparatively decreased in DS and increased in BHS with infection. These data begin to define the interspecies differential immune response to pulmonary M. ovipneumoniae infection in DS and BHS and provide the first investigations of respiratory epithelium-associated IL-17 in ovine.
... Alternative methodologies to the use of animals has led to the development of the 3Rs strategy proposed by Charles Hume in 1954. The postulate of the 3Rs consists of: reducing, refining, and replacing animals in research laboratories (Ranganatha and Kuppast, 2012;Zurlo et al., 1996). Since that time, several ethical, economical and scientific objections against the use of animals have increased in society as a whole (Punt et al., 2011). ...
Article
Alternative models to replace animals in experimental studies remain a challenge in testing the effectiveness of dermatologic and cosmetic drugs. We proposed a model of human organotypic skin explant culture (hOSEC) to assess the profile of cutaneous drug skin distribution, adopting dacarbazine as a model, and respective new methodologies for dermatokinetic analysis. The viability tests were evaluated in primary keratinocytes and fibroblasts, and skin by MTT and TTC assays, respectively. Then, dacarbazine was applied to the culture medium, and the hOSEC method was applied to verify the dynamics of skin distribution of dacarbazine and determine its dermatokinetic profile. The results of cell and tissue viability showed that both were considered viable. The dermatokinetic results indicated that dacarbazine can be absorbed through the skin, reaching a concentration of 36.36 µg/mL (18,18%) of the initial dose (200 µg/mL) after 12 hours in culture. Histological data showed that the skin maintained its structure throughout the tested time that the hOSEC method was applied. No apoptotic cells were observed in the epidermal and dermal layers. No visible changes in the dermo-epidermal junction and no inflammatory processes with the recruitment of defense cells were observed. Hence, these findings suggest that the hOSEC concept as an alternative ex vivo model for assessing the dynamics of skin distribution of drugs, such as dacarbazine, and determining their respective dermatokinetic profiles.
... Commonly used examples are the invertebrates Caenorhabditis elegans and Drosophila melanogaster, and the vertebrates Mus musculus, Rattus norvegicus, Danio rerio, Xenopus laevis and Gallus gallus domesticus [1][2][3]. However, there is current pressure to cut the use of vertebrates, and to use invertebrates instead, to partly satisfy one criterion of the "3Rs" principles (Replacement) [4] in animal welfare [5][6][7]. This requires an evaluation of sentience so that an animal with "higher" sentience is replaced by one that is "lower" (often unprotected by legislation, i.e., embryos of vertebrates before of the last third of their normal development or invertebrates except live cephalopods). ...
Article
Full-text available
Decapod crustaceans are widely used as experimental models, due to their biology, their sensitivity to pollutants and/or their convenience of collection and use. Decapods have been viewed as being non-sentient, and are not covered by current legislation from the European Parliament. However, recent studies suggest it is likely that they experience pain and may have the capacity to suffer. Accordingly, there is ethical concern regarding their continued use in research in the absence of protective measures. We argue that their welfare should be taken into account and included in ethical review processes that include the assessment of welfare and the minimization or alleviation of potential pain. We review the current use of these animals in research and the recent experiments that suggest sentience in this group. We also review recent changes in the views of scientists, veterinary scientists and animal charity groups, and their conclusion that these animals are likely to be sentient, and that changes in legislation are needed to protect them. A precautionary approach should be adopted to safeguard these animals from possible pain and suffering. Finally, we recommend that decapods be included in the European legislation concerning the welfare of animals used in experimentation. View Full-Text
... Current safety testing in all industries is trying to implement the 3R principles which propose the reduction, refinement, and replacement of safety studies performed on animals. [4][5][6] The announcement of EU regulation (EC) No 1907/2006 (widely known as REACH) and the European Union's Regulation (EC) No 1223/2009 (ban on animal testing in the cosmetics industry) shifted the focus from in vivo studies towards in vitro and in silico procedures for risk assessment. Another major step towards this goal was the European Pharmaceuticals Agency's (EPA) announcement of abolishing all funding for mammalian safety testing, as well as to eliminate mammalian safety testing itself from the approval processes by 2035. ...
Article
Full-text available
In silico toxicology is an emerging field. It gains increasing importance as research is aiming to decrease the use of animal experiments as suggested in the 3R principles by Russell and Burch. In silico toxicology is a means to identify hazards of compounds before synthesis, and thus in very early stages of drug development. For chemical industries, as well as regulatory agencies it can aid in gap‐filling and guide risk minimization strategies. Techniques such as structural alerts, read‐across, quantitative structure–activity relationship, machine learning, and deep learning allow to use in silico toxicology in many cases, some even when data is scarce. Especially the concept of adverse outcome pathways puts all techniques into a broader context and can elucidate predictions by mechanistic insights. This article is categorized under: • Structure and Mechanism > Computational Biochemistry and Biophysics • Data Science > Chemoinformatics Abstract How computers can help toxicologists' decision‐making.
... Higher safety standards are important but also require more tests to be conducted. This is contradictory to the 3R principles of reducing animal testing experiments [1][2][3]. Additionally, over the last years pharmaceutical industries have faced a decline in newly marketed drugs [4,5]. The concept "fail early, fail cheap" is gaining increasing importance since every failure in late stages of drug development is associated with high costs [6,7]. ...
Article
Full-text available
Training neural networks with small and imbalanced datasets often leads to overfitting and disregard of the minority class. For predictive toxicology, however, models with a good balance between sensitivity and specificity are needed. In this paper we introduce conformational oversampling as a means to balance and oversample datasets for prediction of toxicity. Conformational oversampling enhances a dataset by generation of multiple conformations of a molecule. These conformations can be used to balance, as well as oversample a dataset, thereby increasing the dataset size without the need of artificial samples. We show that conformational oversampling facilitates training of neural networks and provides state-of-the-art results on the Tox21 dataset.
... Table 2. Intraspecies and interspecies fold change in chromogen deposition in domestic sheep and bighorn sheep without andwith Mycoplasma ovipneumoniae detected in lung tissue.supports the United States legislative mandate to incorporate the three Rs (reduction, refinement, and replacement) into research[23]. Since study design limited the available specimens for BHS to archived FFPE tissue, the opportunistically collected DS lung tissues were similarly fixed and processed. ...
Preprint
Full-text available
Mycoplasma ovipneumoniae is a respiratory pathogen that can impact domestic sheep (Ovis aries; DS) and bighorn sheep (Ovis canadensis; BHS). Experimental and field data have indicated BHS are more susceptible than DS to developing polymicrobial pneumonia associated with Mycoplasma ovipneumoniae infection. We hypothesized that DS and BHS have a differential immunopathologic pulmonary response to M. ovipneumoniae infection. A retrospective study was performed using formalin-fixed, paraffin-embedded (FFPE) lung tissue from DS and BHS without and with M. ovipneumoniae detected in the lung tissue (n=8 per group). While each M. ovipneumoniae positive lung sample had microscopic changes typical of infection, including hyperplasia of intrapulmonary bronchus-associated lymphoid tissue (BALT) and respiratory epithelium, DS exhibited a more robust and well-organized BALT formation as compared to BHS. Immunohistochemistry was performed with antibodies reactive in FFPE tissues and specific for leukocyte and cytokine markers: T cell marker CD3, B cell markers CD20 and CD79a, macrophage markers CD163 and Iba1, and cytokine IL-17. Digital analysis was used to quantitate chromogen deposition in regions of interest (ROIs), including alveolar and bronchiolar areas, and bronchiolar subregions (epithelium and BALT). Main effects and interaction of species and infection status were analyzed by beta regression and Bonferroni corrections were performed on pairwise comparisons (PBon<0.05 significance). Significant species differences were identified for bronchiolar CD3 (PBon=0.0023) and CD163 (PBon=0.0224), alveolar CD163 (PBon=0.0057), and for IL-17 in each of the ROIs (alveolar: PBon=0.0009; BALT: PBon=0.0083; epithelium: PBon=0.0007). Infected BHS had a higher abundance of bronchiolar CD3 (PBon=0.0005) and CD163 (PBon=0.0162), and alveolar CD163 (PBon=0.0073). While IL-17 significantly increased with infection in BHS BALT (PBon=0.0179) and alveolar (0.0006) ROIs, abundance in DS showed an insignificant decrease in these ROIs and a significant decrease in epithelial abundance (PBon=0.0019). These findings support the hypothesis that DS and BHS have a differential immunopathologic response to M. ovipneumoniae infection.
... One reason for this is that we tend to care about animals similar to us, and invertebrates look just very different; invertebrates were considered 'things', not animals, and the discussion as to whether 'lower' animals can even feel pain and suffering is burgeoning (see the online journal Animal Sentience, https:// animalstudiesrepository.org/animsent/).The debate of whether crustaceans feel pain seems never-ending and with rigid positions, yet we need more experiments and data (Elwood 2016;Diggles 2019). Even with the 3R (Refine, Reduce, Replace) animal welfare approach (Zurlo et al. 1996), one of the tenets ('replace') was often considered to be replacement of 'higher' vertebrates with 'lower' invertebrates. Another factor leading to little consideration of the welfare of invertebrates was linked to the underestimation of their behavioural complexity and flexibility. ...
Chapter
This introductory chapter sets up the roots and rationale of the volume while outlining its logical structure, contents and goals. It is about welfare, not of the captive laboratory or farm mammals and birds that we usually consider but about invertebrates, the other 99% of animal species. Invertebrates have been previously relegated to the category ‘things’ with no worry about what we do to them, but new research suggests that their behavioural and neurophysiological complexity was underestimated. Some invertebrates such as cephalopods, crustaceans and insects may feel pain and suffering and may have consciousness and awareness as well. Also, good welfare is going to mean different things to spiders, bees and coral animals. So we aim at taking animal welfare in a very different direction. We start by discussing why we need this book and what the value of non-vertebrate animals might be. The different chapters will focus on specific animal groups, tackling questions that are most appropriate to each one. What is pain in crustaceans, and how might we prevent it? How do we ensure that octopuses are not bored? What do bees need to thrive, pollinate our plants and give us honey? Since invertebrates have distinct personalities and some social animals have group personalities, how do we consider this? And, as in the European Union’s application of welfare consideration to cephalopods, how do the practical regulatory issues play out? This volume provides a first compilation of essays across invertebrate taxa illustrating how and why their welfare should be accomplished and instituted.
Article
Although cisplatin is an effective platinum-based anticancer drug against solid cancer, its availability is limited owing to its adverse side effects. Our study aimed to identify the potential relationship within cisplatin-induced multi-organ physiological changes and genetic factors associated with sex differences in nephrotoxicity susceptibility. To investigate this, mice received a single intraperitoneal injection of cisplatin. Cisplatin administration resulted in renal dysfunction, as evidenced by the elevation in serum biomarkers of renal damage (blood urea nitrogen and creatinine) and the degree of histopathological alterations. In particular, along with testicular damage and low testosterone levels, we also observed a decrease in male-specific (CYP3A2) or male-dominant (CYP2B1 and CYP3A1) CYP isoforms in the livers of rats with hepatotoxicity following cisplatin treatment, which may be associated with an imbalance in male hormone regulation caused by renal and testicular injury. Notably, we found that male rats were more susceptible to cisplatin-induced nephrotoxicity, as characterized by histopathological and biochemical analyses. Therefore, RNA sequencing was performed at baseline (pre-treatment) and at 48 h following cisplatin administration (post-treatment) to identify the genes associated with sex differences in nephrotoxicity susceptibility. Gap junctions, which play a role in replenishing damaged cells to maintain tissue homeostasis, and mismatch repair associated with a pathological apoptotic mechanism against cisplatin nephrotoxicity were significantly enriched only in males following cisplatin treatment. Moreover, among the 322 DEGs showing different basal expression patterns between males and females before cisplatin treatment, the male expressed high levels of genes, which are responsible for transmembrane transport and regulation of apoptotic process, pre-cisplatin treatment; additionally, genes involved in the PI3K-Akt signaling pathway and the oxidation–reduction process were significantly lower in males before cisplatin treatment. Collectively, our comprehensive findings provided valuable insight into the potential mechanisms of sex differences in cisplatin-induced nephrotoxicity susceptibility.
Article
Full-text available
Appropriate education and training of all those engaged in the use of live vertebrate animals far scientific purposes is required by both the Council of Europe (Convention ETS 123, Article 26) and the European Union (Council Directive 86/609/EEC, Article 14). FELASA has elaborated these requirements in practical detail, for this purpose dividing those involved into 4 Categories (although these Categories need not be mutually exclusive): Category A-persons taking care of animals; Category B-persons carrying out animal experiments; Category C-persons responsible for directing animal experiments; Category D-laboratory animal science specialists. FELASA has based its recommendations on functions-which are common to all, rather than on nomenclature-which differs from country to country. The teaching syllabus published here in detail for Category C provides a common basis for other Categories where functions are similar, while the section 'Animal care/husbandry' of Category A Level 1 should satisfy the requirement of Article 14 of the Directive that 'persons carrying out or supervising the conduct of experiments' (Categories B and C) 'shall... be capable of handling and taking care of laboratory animals'. Practical, theoretical and ethical aspects should be treated for all Categories. Refinement of techniques, reduction in the number of animals used and replacement wherever possible by non-sentient systems should be guiding principles. The FELASA recommendations for Categories A and C are presented here. Those for Categories B and D are to follow.
Article
Full-text available
The refinement of experimental techniques represents an important opportunity to improve the welfare of laboratory animals. Objective methods for the assessment of pain and distress in animals are needed before procedures that are claimed to be refinements can be evaluated. The methods currently used for assessment of pain and distress are unsatisfactory, and are often based on uncritical anthropomorphic assumptions. Future developments may enable the establishment of well validated clinical scoring systems, or identification of biochemical or physiological indices of pain or distress. If reliable methods of pain assessment can be developed, then a critical evaluation of the methods available for the alleviation of pain and distress can be undertaken. This article reviews methods of clinical pain assessment in animals, with reference to the techniques used in man. Techniques for pain alleviation are briefly reviewed.
Article
The general standard of statistics in medical journals is poor. This paper considers the reasons for this with illustrations of the types of error that are common. The consequences of incorrect statistics in published papers are discussed; these involve scientific and ethical issues. Suggestions are made about ways in which the standard of statistics may be improved. Particular emphasis is given to the necessity for medical journals to have proper statistical refereeing of submitted papers.
Article
The factors which need to be taken into account in designing a 'good' experiment are reviewed. Such an experiment should be unbiased, have high precision, a wide range of applicability, it should be simple, and there should be a means of quantifying uncertainty (Cox 1958). The relative precision due to the use of randomized block designs was found to range from 96% to 543% in 5 experiments involving 30 variables. However, a survey of 78 papers published in two toxicology journals showed that such designs were hardly used. Similarly, designs in which more than one factor was varied simultaneously ('factorial designs') were only used in 9% of studies, though interactions between variables such as dose and strain of animal may be common, so that single factor experiments could be misleading. The consequences of increased within-group variability due to infection and genetic segregation were quantified using data published by Gärtner (1990). Both substantially reduced precision, but toxicologists continue to use non-isogenic laboratory animals, leading to experiments with a lower level of precision than is necessary. It is concluded that there is scope for improving the design of animal experiments, which could lead to a reduction in animal use. People using animals should be required to take formal training courses which include sessions on experimental design in order to minimize animal use and to increase experimental efficiency.
Article
Poorly designed and analysed experiments can lead to a waste of scientific resources, and may even reach the wrong conclusions. Surveys of published papers by a number of authors have shown that many experiments are poorly analysed statistically, and one survey suggested that about a third of experiments may be unnecessarily large. Few toxicologists attempted to control variability using blocking or covariance analysis. In this study experimental design and statistical methods in 3 papers published in toxicological journals were used as case studies and were examined in detail. The first used dogs to study the effects of ethanol on blood and hepatic parameters following chronic alcohol consumption in a 2 × 4 factorial experimental design. However, the authors used mongrel dogs of both sexes and different ages with a wide range of body weights without any attempt to control the variation. They had also attempted to analyse a factorial design using Student's t-test rather than the analysis of variance. Means of 2 blood parameters presented with one decimal place had apparently been rounded to the nearest 5 units. It is suggested that this experiment could equally well have been done in 3 blocks using 24 instead of 46 dogs. The second case study was an investigation of the response of 2 strains of mice to a toxic agent causing bladder injury. The first experiment involved 40 treatment combinations (2 strains × 4 doses × 5 days) with 3-6 mice per combination. There was no explanation of how the experiment involving approximately 180 mice had actually been done, but unequal subclass numbers suggest that the experiment may have been done on an ad hoc basis rather than being properly designed. It is suggested that the experiment could have been done as 2 blocks involving 80 instead of about 180 mice. The third study again involved a factorial design with 4 dose levels of a compound and 2 sexes, with a total of 80 mice. Open field behaviour was examined. The author incorrectly used the t-test to analyse the data, and concluded that there was no dose effect, when a correct analysis showed this to be highly significant. In all case studies the scientists presented means α standard deviations or standard errors involving only the animals contributing to that mean, rather than the much better estimates that would be obtained with a pooled estimate of error. This is virtually a universal practice. While it is not in itself a serious error, it may lead scientists to design experiments with group sizes of at least 3 animals, which may result in an unnecessarily large experiment if there are many treatment combinations. In conclusion, all 3 papers could have been substantially improved, with higher precision and the use of fewer animals if more attention had been paid to better experimental design.