Table Of ContentCHAPTER 1
Alternative Method of Assessing Toxicity
Chris K Atterwill
1. Introduction
The safety assessment of new chemicals and pharmaceuticals and the
incorporation of these data into a human risk assessmentp ackage requires
a large number of expensive, regulated tests in animal species including,
in some cases, nonhuman primates (I-3).
There are currently a wide range of animal replacement alternative
opportunities in industrial chemical and drug development (Table 1)
(4,.5). Although in vitro methodology has long been used as a basic labo-
ratory tool for defining biological and toxicological processes in differ-
ent cellular systems, application and use alternatives in industrial
compound discovery (i.e., research and development) is slow. Coupled
with a relatively low innovation rate in the design of new in vivo tests for
the toxicological and safety evaluation of new compounds, this has both
ethical and resource implications.
From a basic scientific viewpoint in vitro toxicological models have
made important contributions in elucidating, e.g., the cellular and molec-
ular mechanisms involved in apoptotic and necrotic cell death and in
carcinogenesis and the role of mediators, such as free radicals and
oncogenes, in these processes (6).
The “take-up” of in vitro systems in toxicity testing is, however, now
gradually occurring in industry and resources are being invested slowly
into the area for both ethical and scientific reasons. A lot of emphasis
currently lies on the ethical question as public sensitivity to animal use in
safety testing increases. This has occurred most significantly in the case
From Methods m Molecular Wology, Vol 43 In Wtro Tox/c/ty Testmg Protocols
Edited by S. O’Hare and C. K Atterwlll Copyright Humana Press Inc , Totowa, NJ
1
Atterwill
Table 1
Ammal Replacement Alternatives
Improved storage, exchange, and use of information, so that unnecessary repetition of
experiments on animals could be avoided.
Maximum use of predictions based on physical and chemical properties of molecules
Mathematical modeling of quantitative structure-activity relationships.
Molecular modeling and the use of computer graphics.
Mathematical modeling of biochemical, physiological, pharmacological, and toxlco-
logical systems and processes.
The use of lower organisms not protected by legislation, including invertebrates, plants,
and microbes.a
The use of embryonic and larval vertebrates before they reach the developmental stage
at which time they become protected by law a
The use of in vitro methods including subcellular fractions, perfused organs, tissue
shces, and cell suspensions; and cell organotypic cultures *
Human studies, including epidemiology, postmarketing surveillance, and the properly
regulated use of volunteers.a
% vitro areas
of cosmetic and toiletry safety assessmenta nd the safety testing of chemi-
cal intermediates used in drug synthesis, which have classically used the
controversial Draize, guinea pig, and rabbit tests, and for which valid
alternatives now exist. Furthermore, in drug development the classic LD,,
tests for acute toxicity have not only been partially replaced by the “fixed
dose” procedure, but much work is being carried out by Multicentre
Evaluation of In Vitro Cytotoxicity (MEIC) on new and rapid in vitro
predictors for acute cytotoxicity using human cell lines.
2. Compound Development
Utilizing Alternative Test Models
The development and registration of both new drugs and chemicals
currently requires the submission of a large battery of in vivo toxicity
data derived from a number of species for the “risk-assessment” process
(1-3). The compilation and validation of the animal batteries has been
largely empirical over the years and, although being fairly well-proven
for detecting toxic phenomena in animal species, can have limited pre-
dictive value for human safety assessment for some of the reasons listed
in Table 2 (a,b). There are large lists of chemicals with good animal-
human toxic correlations, but equally, lists of compounds exist that have
been withdrawn from the market because of the increasing number of
clinically reported adverse reactions (ADRs).
Alternative Method of Assessing Toxicity 3
Table 2a
Reasons for Incorrect Predictions from Animal Toxicity Studies
False negative responses
Effect not looked for
Use of inappropriate assay methoda
Improper timing of assay
Insufficient target organ exposure0
Incorrect evaluation of an experimental finding0
Failure to consider absence of preexisting pathological condition
Inability to identify and measure adverse effect0
False negative and false positive responses
Failure to consider differences in metabolic activation and detoxrfrcationa
Disregard of anatomrcal and physiological differences between species“
Inability of animals to express human-specific reaction pattern9
Table 2b
Conversion Factors for Predictmg from Animal Studies to Individuals at Risk
From experiment From humans
Toxic effects to humans! to individuals at risk
Molecular Bioavailability Healthy men and women
Subcellular Pharmacokinetics Babies and children
Cellular Protein bindinga Pregnant women
Tissue Metabolism0 Elderly
Organ Dose and time People at genetic risk
Whole animal Concentrationa Diabetics
Receptor sensitivitya People with infections
Anatomical characteristics Immunosuppressed individuals
Physiological characteristicsa Alcohol or drug abusers
Repau mechanismsa Smokers
Species-specific responsea Patients with organic disease
Mechanisms of actiona Occupationally exposed indivrduals
OAreas in which alternatlves tests can have an impact. (Data taken from table compiled by G
Zbinden, personal communication.)
It is also well accepted that during the development of a “safe” and
effective pharmaceutical (or agrochemical agent) there is a large attrition
rate throughout the safety assessment process with massive financial
implications. When one superimposes on this the varying worldwide
regulatory requirements for administration of a new compound to
humans, one can see a number of important reasons for developing in
vitro toxicity testing systems either as prescreens or as adjuncts to cur-
rent in vivo test packages. This, together with harmonization of the cur-
4 Atterwill
rent regulatory requirements, will hopefully improve the sensitivity and
specificity of animal tests (3).
3. Summary of Gains
1. Financial gains: Reduce attrition rate by use of prescreening strategies prior
to full regulatory animal study packages and develop better predictors of
human toxic phenomena.
2. Scientific gains: Describe more effectively the lesions seen m vivo from
regulatory studies and give better definition of safe concentration and
clinical dose. Define “direct toxicant effects” on target organs as opposed
to indirect effects, and give human reaction mdication using primate
cells. “Adjunct” studies will improve sensitivity and specificity of ani-
mal studies.
3. Ethical gains: Implementation of 3R strategy (reduce, refine, replace am-
ma1 testing). Supplement and reduce current m vivo toxicity tests, particu-
larly those involving distressing procedures and the use of large mammals
and primates.
4. Principles, Aims,
and Types of In Vitro Toxicity Testing
The general advantages and disadvantages of in vitro testing for toxic-
ity are described in Table 3.
5. Validation
The successful use and industrial and regulatory acceptance of a new
in vitro test model depends on a certain degree of validation (7,s). Detailed
validation is generally required if the in vitro test is to replace an in vivo
test or is to be used as a prescreen where financial factors are critical.
However, when the result from an in vitro procedure is submitted to a
regulatory authority along with that from an in vivo test package in order
to explain a lesion, then full validation is not formally necessary as long
as good laboratory practice (GLP) procedures have been adopted in the
execution of that test and the test and endpoints have acceptable rele-
vance. For example, the gradual replacement of the Draize procedure by
tests such as the EYETEX or SKIN2 tests has required extensive valida-
tion of that test. So, for example, would the use of a prescreen for a new
immunosuppressant with adrenal toxicity where there were a limited
number of available backup compounds or they were very expensive to
synthesize. On the other hand, if a drug company were trying to confirm
to the Food and Drug Administration (FDA) that a particular drug had no
Alternative Method of Assessing Toxicity 5
Table 3
Advantages and Disadvantages of In Vitro Systems for Detection
of Xenobiotic-Mediated Toxicity
Advantages (general)
Detect direct (vs Indirect) toxrc/cytotoxic effects on target organ.
Use controlled conditions of exposure-concentration of toxrcant known.
Study parent compound vs metabolite (rt liver S9 metabolizing fractions from
different species)
Study effects on cells vs subcellular organelles.
Has resource implications (time, animals, number of compounds tested).
Disadvantages (general)
Systems not always representative of mature, differentiated target organs (cells
dedifferentiated in cell lines?).
Xenobiotic concentrations not representative of those in vivo (e.g., plasma
protein bindmg factors).
Biological barriers absent (e.g., blood-brain barrier in neural cultures of CNS).
Metabolite profiles differ
Difficulty of culturing/maintaining certain target organs in vitro.
direct neurotoxic effect in humans despite some minor behavioral
changes detected in the rat, then submission of data from cultured human
exposed neurons would be acceptable, probably without full validation
of that particular culture model.
Accepted validation criteria for an in vitro system are described in
Table 4 and include definition of the specificity, sensitivity, and predic-
tive value of such a test. The validation parameters are obtained by con-
ducting blind validation trials. It is my belief that the requirements for in
vitro test validation can be summarized as follows:
1. Full validation involving multicenter coordination: To support in vrvo test
replacement.
2. In-house validation: In vivo test reduction, to support,e .g., development
of a prescreen,
3. Limited inter- or intralaboratory: To supportr efinement or supplementa-
tion of in vivo toxicity test data, to develop adjunct tests;u se of the model
to define basic scientific toxic phenomena.
It is believed that validation should not be used as an excuse for
nonadoption, nondevelopment, or nonacceptance of in vitro methodol-
ogy. Sadly, and largely for political reasons, this scenario still exists in
many companies and countries.
Atterwill
Table 4
Validation Criteria for In Vitro Test Models
A formal validation study will require:
Careful selection of chemicals (mimmum 20-40?)
Use of chemical pairs
Toxicological classification from m vivo data
“Blind” testing to be performed
Method for evaluation of test outcome (absolute values)
Method for evaluation of test performance
Methods of expressing test performance
Other points
Are there good in vivo comparative data for compounds chosen?
Which kind of in vivo assay trying to emulate/evaluate in vitro?
Agree with collaborating centers in validation trial at beginning who
will be organizing and collatmg data.
6. Spectrum of Available In Vitro Toxicity Tests
The currently available models in In Vitro Toxicology (Table 5) (5-8)
span six main areas: reproductive toxicity, mutagenicity, irritancy test-
ing, immunotoxicity, target organ toxicity (including endocrine and neu-
rotoxicity), and ecotoxicity involving the use of fish, invertebrates, and
so on. Within these main areas there are also important subareas.
As alluded to above there are various modes in which to operate these
tests in an industrial setting and generally the mode predominance varies
significantly according to both scientific area and whether or not one is
operating in the drug, chemical, or cosmetic industry. For example, a test
system might progress from unvalidated use in the fine description of a
pathologically identified lesion for a lead development compound, to the
subsequent, semivalidated use of this system in a prescreening mode for
second-generation drug candidate compounds. Alternatively, the agro-
chemical industry has developed a tiered in viva/in vitro hierarchical
model for the labeling of industrial chemicals as skin irritants. This latter
development was performed under the auspices of the British Toxico-
logical Society, showing how the scientific and industrial communities
can interact so well on such issues. Here, a chemical for irritancy classi-
fication would proceed from tests on isolated skin or cells in vitro to tests
in a limited number of animals in vivo depending on negative or positive
outcomes in the initial in vitro tests.
Alternative Method of Assessing Toxicity 7
Table 5
In Vttro Models Currently Available in Toxicology
Mutagenicity testing
Irritancy testing
Reproductive toxicity testing
Quality-Structure Activity
Relationship (QSAR)
Target organ toxicity
Immunotoxicity
Hemtc system
Endocrine toxicity
Neurotoxtctty
Acutelcytotoxicity testing
7. Recent Successes
and Developments in In Vitro Toxicity Testing
It is refreshing to observe the momentum that is now gathering in this
field (see Table 6) and the way in which “in the face of adversity” some
tests are being accepted as full replacement alternatives. It is noteworthy
to say that a lot of this energy has been provided by the public and by
academic research centers.
Apart from the mutagenicity test area where many innovations con-
tinue to occur, some of the following recent developments in other areas
warrant attention.
1. Eyetex, Skmtex, and Corrosrtex tests for eye and skin irritancy (Ropak
Corporation Ltd) and the SKIN2 Model (Advanced Tissue Sciences). More
recently, the Ropak Solatex system for predicting photoirritation in
vitro.
2. The use of hepatocyte “couplets” for in vitro investigation of xenobiotic
effects on bile flow. Together with measurements of hepatotoxicity and
fatty acid accumulation by these cells, rt may now be possible to obtain a
complete hepatoxicological profile in one in vitro model.
3. Luminescent bacteria (Microtox test) for measuring the ecotoxic potential
of industrial effluent.
4. More sensitive in vitro toxicity measurements using, for example, the mito-
chondrial MTT test for succurate dehydrogenase activity. This test gives a
more sensitive and earlier prediction of toxicity than classical LDH or neu-
tral red measurements.
Atterwill
Table 6
Orgamzatlons Involved m the Development of Alternative Testing
Bodies for promotion of alternatlve nonanimal testing
FRAME-Fund for Replacement of Animals in Medical Experiments
ERGAT-European Research Group for Alternative Testing
EURONICHE-European group for alternatlve methods for biology teaching
CAAT-Center for Alternatives to Animal Testings (Johns Hopkins Medical
School, Baltimore, MD)
Dr. Hadwen Trust-Nonammal research and testing strategies (UK-based)
Societies, conferences, and journals advancing alternative testing
PIVT-Practical In Vitro Toxicology conference
IVTS-In Vitro Toxicology Society (UK)
Scandinavian Cellular Toxicology Society
FRAME Toxicity Committee and Conference
TIV-Toxicology In Vitro, Journal
ATLA-Alternatwes to Laboratory Animals, Journal
Hildegard Doerenkamp and Gerhard Zbinden Foundation for Reahstlc Animal
Protection and Scientific Research, Switzerland
5. Measurements of calcium accumulation in single cultured neurons for the
measurement of neurotoxicity.
6. Tlered tests involving both simple and organotyplc organ systems; hierar-
chical proceeds involving a battery of m vitro and in vivo models.
8. Conclusions
Industry and academia have come far in developing in vitro alterna-
tives, and bodies such as Fund for Replacement of Animals in Medical
Experiments (FRAME), European Centre for Validation in Alternative
Methods (ECVAM), and Center for Alternatives to Animal Testing
(CAAT) (USA), have simultaneously enhanced public and regulatory
awareness (Table 6). The regulatory and industrial acceptance of new
alternative tests depends on proper, well-coordinated validation trials at
a level befitting the intended use of the alternative test. This has started
through FRAME and European Community (EC) initiatives and good
examples have been set by the cosmetics and toiletry industries. More
commercial “takeup” is still required for these new tests at the toxico-
logical prescreening and in vivo adjunct testing level. Regulatory har-
monization of in vivo animal testing is occurring for both ethical and
resource reasons. The gradual replacement of the LD,, test by the “Fixed
Alternative Method of Assessing Toxicity 9
Dose” procedure for acute toxicity testing and the realization that 6 mo
chronic testing (I) is sufficient to identify the most important pathology
(excluding carcinogenicity) are most welcome changes. The potential
risks for humans in adopting alternative toxicity tests are few and ben-
efits great if the data generated is used correctly. The imminent replace-
ment of all in vivo tests is unlikely but in the future may gradually occur.
In the meantime in vitro tests will continue to supplement the somewhat
“impirical” animal tests for human toxicity.
References
1. Volans, G. N., Sims, J., Sullivan, F. M., and Turner, P. (eds.) (1989) Basic Science
in Toxzcology, V International Congress of Toxicology (ICTV), Taylor & Francis.
2 Poole, A. and Leslie, G. B , eds. (1989) A Practical Approach to Toxological
Investigations, Cambridge University Press.
3. Lumley, C., Parkmson, C, and Walker, S. R. (1992) An international appraisal of
the minimum duration of chronic animal toxicity studies. Hum. Exp. Toxicol. 11,
155-162
4. Parish, W. E. and Hard, G. C. (eds.), Toxicology In Vitro. Proceedings of Second
International Conference on Practical In Vitro Toxicology 4/5.
5. Atterwill, C. K. and Steele, C. E., eds. (1987) In-Vitro Methods in Toxicology,
Cambridge University Press, Cambridge.
6. Walum, E., Stenberg, K., and Jenssen, D. (1990) Understanding Cell Toxicology-
Principles and Practice, Ellis Horwood, New York, London, Sydney.
7. OECD Environment Monograph No. 36. Scientific Criteria for Validation of In
Vitro Toxicity Tests, Sept. 1990.
8. FRAME 21st Anniversary Issue (1990) ATLA, 18.
