226 Scandinavian Cell Toxicology Congress 11

Validation ofIn Vitro Cytotoxicity Tests - Past

and Present Strategies

Bjorn Ekwall 1, Inger Bondesson", Sven Hellberg3, Johan Hogberg", Lennart

Romert'', Kjell Stenberg" and Erik Walum7

'Department of Toxicology, Biomedical Center, University ofUppsala, Box 594, S-751 24

Uppsala, Sweden;2SwedishPoisonInformation Center, KarolinskaHospital,Box60500,S-104
01 Stockholm, Sweden; 3Research Group for Chemometrics, Department ofOrganic Chemistry,
University of Umea, S-901 87 Umea, Sweden; "National Institute of Occupational Health,
Ekelundsoiigen. 16, S-171 63 Solna, Sweden; 5Department ofGenetic and Cellular Toxicology,
Wallenberg Laboratory, University ofStockholm, S-1 06 91 Stockholm, Sweden; "Department
ofMedical Radiobiology, KarolinskaHospital, S -10401 Stockholm, Sweden; 7Department of
Neurochemistry and Neurotoxicology, University ofStock holm , S -10691 Stockholm, Sweden

Summary - In recent years, conventional toxicity testing in animals has been reinforced by
in vitro methods. As a result, toxicity testing in some sectors has become more effective and at
the same time more ethical. This trend is probably only at its beginning, as many of the
newly-developed methods have not yet won general acceptance as a basis for the large-scale
replacement and reduction of animal experimentation. What limits the wider use of these
methods is validation, i.e. the evaluation of their reliability and relevance. The present paper
is a short review of the validation efforts made hitherto, including projects being planned and
under discussion. Our own MEIC approach is compared with other strategies. Finally, our
opinion on the effectiveness of one large consensus project relative to several different smaller
validation programmes is expressed - we advocate the latter strategy, because it will save time
and reduce costs.

Key words: cytotoxicity assays, evaluation, toxicity testing, validation.

Introduction actual performance of validation has been

Progress in the in vitro testing of general
(non-genetic) toxicity depends on the
development of new methodology, as well as
on evaluation of the relevance and reliability
of the new methods. The latter task is
commonly called validation, and seems to be
more difficult than method development.
Thus, several hundred different in vitro
assays of general toxicity have been proposed
in the last twenty years, but only very few of
them have been validated and accepted. It is
generally believed that validation is the key
to extending the use of in vitro methods as
supplements or alternatives to the animal
testing of the systemic, target organ and local
toxicities of chemicals. Since the recognition
of validation as a key process in the early
1980s (1, 2), the speed and efficiency of the
modest. However, in the last few years, new
strategies have been proposed and discussed
(3-5) in parallel with the practical start of
many new programmes (6-12).
Single Laboratory Validation
The mere description of a new in vitro test,
even including in vitro/in vivo toxicity
comparisons for 10-20 chemicals, cannot be
considered as validation. However, some
developers of tests have passed that stage and
performed validation of their methods by
testing and comparing a larger number of
chemicals (e.g, 50-100), including
collaboration with other laboratories in blind
studies for evaluation of the method's
interlaboratory variability. In some
Scandinavian Cell Toxicology Congress 11
instances, such single laboratory validation
has led to the practical use of the method in
industry. Recent interest in multicentre
programmes must not make us forget the
contribution of such validation to
achievements in the field, some examples of
which will be presented in the following
paragraphs.
Local irritancy of implants
As early as 1965, Rosenbluth et al. (13)
demonstrated that a mouse cell line toxicity
assay resulted in a reasonably good prediction
of the irritancy ofintramuscular implants and
injections in rabbits. In their study,
qualitative (+/-) in vitro/in vivo comparisons
were made for 112 samples (13). Many false
positive results were eliminated when an agar
overlay modification of the test was used (14).
A later qualitative validation study with 1013
materials confirmed the earlier findings (15).
As a result, cellular toxicity assays have for
a long time been accepted as routine
supplements in biomaterials testing.
Screening of acute systemic toxicity
One of the laboratories in our group has
performed a series of validation studies on
the prediction of acute human toxicity by a
HeLa cell assay (summarised in [2]). The
cytotoxic concentrations of 50 non-selected
drugs to HeLa cells were compared
quantitatively with human lethal and toxic
blood concentrations, including a study of
lethal and toxic symptoms. Surprisingly, it
was found that the HeLa cell results
(representing basal cytotoxicity) were
relevant for the target organ toxicity of a
majority of the drugs. Further evaluation,
with the use of other cell types (16) and other
classes of chemicals (17), supported the
finding that simple cytotoxicity assays with
cell lines are very often predictive of acute
systemic toxicity, provided that allowance is
made for the toxicokinetics ofthe chemicals.
Evaluation of the correlation between
cytotoxicity and acute lethal dosage for
rodents has also been performed (17-22).
Typically, 25-50 chemicals have been used in
quantitative in vitro/in vivo comparisons with
a resulting linear correlation of about 0.75.
This correlation probably corresponds to an
even better correlation between toxic
concentrations to target cells in vitro and in
vivo, which cannot be demonstrated in the
absence of biotransformation and other
227
toxicokinetic data (18, 21, 22).
These validation studies have led to the
widespread acceptance of in vitro cytotoxicity
tests by industry (23), as so-called prescreens
of acute toxicity for use during product
development as well as a vital part of the
detailed investigations of pharmaceuticals
(24, 25), to be conducted along with animal
tests, toxicokinetic studies and
biotransformation studies (often carried out
on human hepatocyte cultures).
Eye irritancy
As a result of animal rights campaigns in the
early 1980s against the performance of the
Draize eye irritancy test in rabbits, academic
scientists and industry in the US and in
Europe have focused their attention on the
development and validation of in vitro eye
irritancy tests.
One group developed a battery of tests,
including a neutral red uptake assay and a
protein assay for measuring cell viability and
cell proliferation, and performed intra-
laboratory and interlaboratory evaluation
with several classes of chemicals (26). The
predictive value of the tests was evaluated by
determining correlations between in vitro and
Draize test data. The choice of the Draize test
score as the endpoint of evaluation may be a
handicap, since it is somewhat arbitrarily
composed of several measurements of injury
in the rabbit eye, which are of doubtful
relevance to concentration- and time-
dependent quantitative human eye irritancy.
Separate external studies of a total of 150
coded substances generally resulted in rank
correlations of 0.5-0.6. When all classes of
chemicals were considered together, the
correlations were not as good as those
obtained for each separate class.
Another laboratory has developed and
validated the CAM test, utilising the chorio-
allantoic membrane (CAM) of hens' eggs as
the test material (27). An improved CAM test
was challenged with 15 products, with a
resulting rank correlation with Draize test
scores of 0.6-0.8 (28). A modification of the
test has been developed in Germany - the
Hens Egg Test/Chorio-Allantoic Membrane
(HET/CAM) test (29). According to
developers, a validation study of250 products
demonstrated good relevance and reliability,
but raw data were not presented (30). Other
laboratories have failed to obtain such good
results (30, 31).
228
Arguably the best validated in vitro test
for eye irritancy at present is EYTEX™, a
simple protein-aggregation test which
involves the use of a protein matrix based on
jack bean powder (32). A 90% correlation with
Draize test data was obtained for 130
substances and materials tested externally by
12 industrial companies.
A two-laboratory double-blind study of 49
products demonstrated the same predictive
value and also a 93% agreement of results
from the two laboratories (33). A database of
over 1500 tested products has been reported
(34). When EYTEX™ and the neutral red
uptake assay were compared in a recent study
of 17 solvents, both predicted the corneal
opacity score of an EEC modification of the
Draize test equally well (r=0.84; 35). To some
extent, the good correlation between
EYTEX™ and Draize test scores can be
explained by the heavy weight of the corneal
opacity score in the total Draize score, corneal
opacity probably being the result of protein
denaturation by chemicals.
The single-laboratory validation exercises
described have already led to the practical
use of all the above-mentioned methods by
industry. In all cases, the validation process
has resulted in effective modifications of
original methods. For example, the original
EYTEX™ method has been modified to a
membrane partition assay, to cope with
insoluble substances (34).
Multi-laboratory validation studies
Multicentre studies have several advantages
over single-laboratory validation studies (1,
2). Firstly, the credibility of results will
increase if an independent group, not itself
involved in the cytotoxicity assays, has
selected the chemicals for further blind
testing among the participating laboratories.
Secondly, the work ofgathering, selecting and
standardising the in vivo toxicity data is
reduced. Thirdly, only a multicentre study
can directly evaluate the predictivity of a
combination of methods. Fourthly, once the
standard procedures for validation have been
determined, the relevance of an unlimited
number of methods can be evaluated by use
of computer techniques. Thus, no potentially
valuable method has to be excluded from an
evaluation of relevance. Finally, inter-
laboratory reproducibility studies are
facilitated by this approach. In the following
sections, all the on-going and proposed
Scandinavian Cell Toxicology Congress 11
multicentre programmes known to us will be
discussed.
The FRAME programme
The first multicentre vaidation programme
was initiated in 1982 by FRAME (Fund for
the Replacement of Animals in Medical
Experiments; 1). One of the first parts of the
programme was to develop a reliable cell line
toxicity assay to measure fundamental
(basal) cytotoxicity. This was done in
cooperation with four laboratories, resulting
in the FRAME ken acid blue (KB) assay (36).
Reliability was assured by extensive multi-
laboratory testing of coded substances. A
recent, similar project has been to develop a
sensitive eye irritancy assay, resulting in a
neutral red release test, with a short exposure
period of one minute (37).
A second part of the programme extended
the testing of the coded substances to
international laboratories. The primary goal
was to evaluate the reliability of the various
methods, but relevance was also evaluated
by comparison of in vitro data with toxicity
profiles of some of the 150 chemicals (36). The
correlation between the FRAME KB test and
rat LD50 values was found to be good (20).
Recently, one international laboratory
compared their Hep-2 cell results with four
British assays (38). Cytotoxicity was similar
in all the systems, independently of the types
of cells and toxic endpoints employed,
supporting the basal cytoxicity concept.
FRAME has emphasised the many possible
pitfalls of multicentre programmes (39, 40).
Among the lessons learned were that good,
and preferably human, toxicity data should
be secured for the reference substances and
that general cut-off points f~r the cytotoxicity
data should be avoided.
SDA and CTFA validation programmes
Two US industry associations, the Soap and
Detergent Association (SDA) and the
Cosmetic, Toiletry and Fragrance Association
(CTFA), have organised evaluation
programmes for eye irritancy tests. The SDA
programme consisted of two phases (7, 41),
In phase I, invited laboratories tested 8 coded
substances in 14 cellular assays, to sort out
the best methods. In phase II, 15 coded
materials were used to challenge nine
candidate systems (two CAM modifications,
corneal plasminogen activator assay with
Scandinavian Cell Toxicology Congress 11
rabbit corneal cells, SIRC/cloning efficiency
of rabbit corneal cells, the uridine uptake
assay, a cell protein accumulation assay, the
neutral red uptake assay, Tetrahymena
motility assay, and chromium release from
murine mastocytoma cells). The methods
generally could discriminate between
materials indicated by the Draize test to be
irritants and non-irritants. An algorithm of
pH and cytotoxicity was needed for
discrimination, rather than cytotoxicity
alone. The CTFA has started their prog-
ramme (8). The phases of the CFTA
programme correspond to evolution of
different classes of chemicals, in Phase I, ten
alcohol-containing generic prototypes from
the cosmetic industry have been tested in 25
in vitro assays.
Other on-going multicentre programmes
A programme was. started. in. 1988 ~n
Germany, with the aim ofvahdatmg tw.o m
vitro tests for eye irritancy (a combmed
neutral red uptakelKB cytotoxicity assay and
the HET/CAM assay; 9, 42). The study is
similar to the Amden protocol (vide infra; 5).
Firstly, the reliability of the methods will be
determined by an intralaboratory and
interlaboratory study involving 14 contract
laboratories testing 35 chemicals. Later, an
in vitro database with 200 chemicals will be
established to be used in the evaluation of
predictions' from non-animal tests against
Draize test scores. So far, an acceptable
reproducibility of both tests has been found,
as well as a similar tendency of the tests to
produce false-positive resul~s. . . CAAT has proposed an outline of future
In France a similar validation of two m
vitro tests for eye irritancy has started, with
use of 40 substances (10). In the same
country, an interesting multicent~e
validation of in vitro tests for acute hepatic
toxicity/hepatic metabolism has also been
performed (12). Six laboratories tested 30
compounds in assays using primary cultured
rat hepatocyte or a rat hepatoma cell line, at
a set exposure time of 20 hours. Most of the
compounds tested were equally cytotoxic ~o
both cell types, indicating a basal cytotox~c
effect. Some specifically hepatotoxic
compounds were relatively more toxic to ~he
primary cultured rat hepatocytes, confirmmg
the results from a previous study of
differential cytotoxicity in hepatocytes and
cell lines (16).
229
Proposed programmes
In 1986, the Johns Hopkins Center for
Alternatives to Animal Testing (CAAT)
arranged a conference entitled Approaches to
Validation. One keynote speaker preferred a
sensitive and flexible validation process
rather than a detailed road-map leading
inevitably to the hidden prize (43). He pointed
out that models in some cases might make
predictions on a functional basis, without the
necessity to be mechanistically defined. A
second introductory speaker emphasised that
mutagenesis test validation has shown that
a high correlation for single endpoint tests
will probably not be achieved, since multiple
mechanisms are often involved in the lesion
of interest. Therefore, validation cannot be
based on a unifying concept (44). The latter
speaker also advocated the use of human,
clinical data for validation purposes.
A third US industrial toxicologist recently
outlined a collaborative validation of
candidate in vitro eye irritancy tests (4): a
committee should select two panels of a
representative variety of test compounds with
known human and rabbit eye irritation (20
and 30 materials, respectively). All
investigators with candidate systems should
be invited to test the coded panel A
compounds. After the data obtained had been
returned, the systems could be evaluated ~s
"go" or "no go", based on results and economic
factors. Finally, the "go" systems would test
the panel B compounds, and this could result
in identification of the best single tests and
test batteries, which later could be
recommended for use in industry.
validation of in vitro toxicity tests (3), in part
built on a previous model (45). Validation is
visualised as having four phases: phase I
includes the preliminaries, such as definition
of the objective of each candidate test; phase
II is a microvalidation of 20 substances,
performed in the primary laboratory; p~ase
III is a macrovalidation of 50-100 chemicals
performed in a contract laboratory, followe.d
by statistical evaluation; and phase IV IS
battery optimisation. All the testing is
performed with coded substances. Th~ e~~ly
feasibility study is focused on reliability
evaluation, while macrovalidation is focused
on evaluation of the relevance of the methods
involved. This outline has been comp-
rehensively expanded in a subsequent OECD
document (46).
230
In February 1990, European and American
cell toxicologists held a workshop in Amden,
Switzerland, with the aim of preparing a
consensus document defining the proper
performance of large-scale validation of in
vitro cytotoxicity tests (5). The outlined
scheme resembles earlier FRAME (1,36) and
CAAT (3, 41) proposals, and the major steps
of validation are defined as:
1. Intralaboratory assessment;
2. Interlaboratory assessment;
3. Test database development; and
4. Evaluation.
Validation is performed with four sets of
coded reference chemicals from a database
set consisting of 200-250 chemicals. The first
steps are mainly concerned with evaluation
of test reliability, except for some relevance
evaluation in the first step with 20-50
chemicals. Then the ultimate relevance of a
method is determined by an evaluation
following the test database development. One
typical feature of the proposal is that
developing laboratories have a rather minor
role in the validation process. The programme
is built on coded substances, contract
laboratories and decision-making by several
expert panels. Thus, decisions by a panel are
crucial to the acceptance of the methods to
be validated. Another typical feature is that
relatively trivial aspects of future validation,
such as evaluation of the variability and
reproducibility of tests, integration of the
validation process, battery selection and
implementation, are discussed extensively.
The likely tricky part, i.e, how to properly
evaluate the relevance of non-genotoxic in
vitro tests, is based on either previous
qualitative (+/-) genotoxicity validation
studies or the previous, debatable, semi-
quantitative validation of in vitro eye
irritation tests against Draize test scores.
Thus, there is no consensus in the protocol
on how to evaluate methods with the use of
human data, toxicokinetic algorithms, and
other probably more relevant approaches.
The MEIC programme
The Scandinavian Society of Cell Toxicology
started a multicentre evaluation of in vitro
cytotoxicity (the MEIC. programme) a few
years ago (6). In the spring of 1989,
international laboratories willing to take part
were invited to test a list of 50 reference
chemicals using their various in vitro assays.
Scandinavian Cell Toxicology Congress 11
All the reference chemicals have data on
acute human toxicity, including toxico-
kinetics, plus rodent oral LD50 values. Many
of them also have other human or animal
data, e.g. on irritancy in the eye and skin.
Another 50-100 chemicals will be added to
the list. Incoming results will be evaluated
on their relevance for various types of human
toxicity (acute and chronic systemic toxicity,
target organ toxicity, local irritancy). The
evaluation will be quantitative and linear. It
is presumed that results from the cytotoxicity
tests, including in vitro tests with
extracellular targets, must be inserted in
toxicokinetic models to be fully predictive for
both systemic toxicity and local irritancy. To
be able to judge the effectiveness of in vitro
tests, prediction of human toxicity by
conventional animal tests for the same
chemicals will be evaluated in parallel. Blind
testing will be achieved by the majority of
participants, although we have not used
coded substances in the first, major phase of
the programme. In a second, final phase, the
tests with the best prediction of human
toxicity will be evaluated for reliability by
contract laboratories and with coded
substances (if they were not known to be
reliable before). The aim is to be able to
recommend tests and batteries of tests as
supplements or alternatives to animal testing
for various types of general toxicity. The
selection of test batteries will be based on soft
multivariate modelling (47).
At present, 62 laboratories have joined the
project. Twenty laboratories have returned
results from more than 50 assays for the first
10 chemicals. Methods for evaluating acute
lethal toxicity (48) and acute sublethal
toxicity (49) have been worked out with the
first incoming results as training sets. Models
for evaluating other types of toxicity are in
progress. To facilitate evaluation of eye and
skin irritancy, we will try to obtain data on
quantitative minimal irritancy in man.
Another optional part of the programme is to
encourage participants to develop key
methods which are missing at present, such
as long-term exposure cytotoxicity assays.
MEIC is essentially an economical and
practical way for a laboratory to extend its
own "single-laboratory" validation of a
method on its own terms. The laboratory has
to finance its own contribution, but the profit,
in the form of data from other methods and
the evaluation against human data, is large
compared with the investment. Thus, the
Scandinavian Cell Toxicology Congress 11
expenses of the central operations can be
minimised. Further, open entry to the
programme will ensure that few potentially
valuable methods will be missed, for instance,
by too early a judgement of their supposed
qualities. This is achieved by beginning the
programme with the relevance phase of
validation. To end the study with the
reliability phase also minimises costs - this
costly part of the study (contract laboratories,
coded substances) can be cut down by the
previous exclusion of clearly irrelevant
methods.
Comments on the Best Way to Validate
It is clear that more effective and
comprehensive efforts for validating in vitro
toxicity tests are needed. These efforts should
probably be of the multicentre type, using
refined and relevant evaluation methods.
Otherwise, relatively ineffective in vitro
methods may be accepted by industry and
regulatory agencies on the merits of vigorous
single-laboratory validation only, at the
expense of potentially more valuable methods
which have not yet entered any validation
contest.
However, we do not believe that it is
necessary to reach a worldwide consensus of
the outline of an ultimate validation process.
Probably such a guideline will have flaws and
biases, like every project looked at in
retrospect (compare the FRAME self-
criticism [39]). Instead, there is an advantage
with a series of different and smaller "local"
programmes. After consideration of the
different angles (aims, methods, flaws, etc.)
of a series of programmes, the common
features of the results of the various studies
will be accepted as valid by the scientific
community, industry and regulatory
agencies. Moreover, a traditionally split
scientific "market", i.e. several different
multicentre validation studies, will allow the
developers of tests a choice of validation
schemes according to their specific needs -
a method could, for instance, be evaluated for
relevance in a MEIC-type study, and at the
Harne time be evaluated for reliability in an
Amden-type study.
If industry and governmental organ-
isutions, such as the EC and the OEeD,
favour the idea of a universal consensus
strategy, like the Amden protocol, it will take
time to reach agreement of the critical parts
231
of the programme not yet discussed (e.g. how
to perform an adequate evaluation of the
relevance of tests), and it will ultimately cost
a lot of money, if the basic strategy of the
protocol is still adhered to at that time. A
better policy would be to fund on-going single-
laboratory and multicentre projects of various
kinds directly, as well as initiating less
ambitious, but more practical, projects, such
as the eye irritation validation process
proposed by Gad (4).
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