Sometimes cell cultures are not sufficient for toxicology
investigations
In animal experiments, the principle of the "3Rs" applies:
Replace, Reduce and Refine
According to the Swiss Animal Protection Act (Tierschutzgesetz,
Article 12), an animal experiment is "... any measure involving
the use of live animals designed to test a scientific assumption,
obtain information, isolate or test a substance or ascertain
the effect of a specific measure on the animal, as well as
the use of animals for experimental behavioural research...".
Great efforts are currently being made to reduce the number
of laboratory animals used in experiments as far as possible,
not just on ethical grounds, but also for scientific reasons.
In this connection, industry and research are focusing on
the strategy of the "3Rs", with the keywords Replace (replace
animal experiments as far as possible by pain-insensitive
systems or compensate by using methods that do not involve
laboratory animals), Reduce (develop methods so that fewer
laboratory animals need to be used) and Refine (improve methods
so that animals undergo less discomfort during the experiments).
1. Animal experiments from antiquity to the present day
Even hundreds or thousands of years ago in Ancient Egypt
and China, some healers were concerned not just with illness
and health in humans, but also in animals. Even in those days,
experiments were systematically carried out in order to help
them gain a better understanding of the biological and medical
background involved. The Greek physician Galen, born
in Pergamon and recognised as the founder of physiology, acquired
much of his knowledge in the 2nd century BC from experiments
with monkeys and pigs. Although he located the relevant areas
as being in the ventricles of the brain, he ascertained, for
example, that the brain was the centre of voluntary movement
and sensation. Animals were also used for investigating physiological
relationships. When Jan de Wales, around 1640, alternately
applied pressure to the large artery or vein in the thigh
of a prepared dog carcass, he observed how the vein emptied,
whereas the artery became swollen. He thus managed to obtain
experimental proof of the link between the vascular system
of arteries and veins. Various vaccines for humans were also
developed with the aid of animal experiments, for example
the vaccine against diphtheria using guinea-pigs and those
against yellow fever and polio using mice and monkeys.
For some years now, transgenic animals have been used
in all areas of biomedical research. As "customised" models
of human illnesses, they supplement the traditional research
methods wherever an illness can be associated with one or
more genes. They help us obtain a better understanding of
the relevant illnesses, develop new treatments and test their
efficacy.
Research has to resort to animal experiments in many cases.
In accordance with animal protection legislation, the researcher
must try to use as few laboratory animals as possible and
subject them to the least possible suffering and pain. This
is not just an ethical requirement, but arises from the directions
of the legislator. It also accords with industry's commercial
objective: since animal experiments are time-consuming and
expensive, substitute methods can frequently lead to cost
savings.
2. A living organism is more than the sum of its individual
cells
Tests on simple systems such as cultures of bacteria, cells
or isolated tissue samples, can provide valuable information
on many questions in toxicology. They are particularly suitable
in comparative investigations for filtering out (screening)
similar substances with similar sites of action, and can provide
specific insights that often remain undiscovered due to the
complexity of the metabolic reactions in a complete organism.
Nowadays, cell cultures are used primarily in the areas of
blood disorders or reproduction toxicology.
In order to be able to test the action of antithrombotic substances
in a blood vessel, for example, scientists used to have to
artificially create a plug (thrombotic occlusion) in the animal.
This would involve damage to the ear vein of a rabbit. But
these days such anticoagulants can be analysed, and a quicker
result obtained, in a test tube using donated human blood
as the starting material. Similarly, phototoxic substances
are no longer applied to shaven areas of rabbit skin and exposed
to light, as used to happen in years past. Rather, they are
detected in connective tissue cells taken from mice and placed
in culture. Test substances in various concentrations are
applied to the cells and the trial setup is irradiated with
UV-A light. A reference substance is left in the dark. After
about 24 hours, the samples are stained, enabling the investigator
to determine how many cells have been damaged or died.
However, since cell cultures are simply accumulations
of similar cells in a nutrient solution, they only show limited
metabolism. Quantitative data on the toxic dose for the whole
body can rarely be derived from such studies, nor is it possible
to determine the target organs of the active drug, or the
symptoms or the profile over time of a harmful effect.
Similarly, effects arising from the interplay of various
organ systems or tissues in the whole body cannot be simulated
using cell cultures, not to mention pain, narcotic effects,
degeneration of certain sections in the central nervous system
or other phenomena such as blood pressure, fever, swellings
or intestinal movements. The same also applies to the simulation
of various reaction patterns of different animal species or
sex-specific reactions in the same species, either of which
might be crucially important for the reliability of a toxicological
risk assessment.
3. Statistics confirm the use of fewer animals
Thanks to the above-mentioned "3Rs" principle, the number
of animal experiments requiring a permit have shown a steady
decline in recent years in Switzerland, as in most EU countries:
for the 16th time in succession according to a bulletin issued
by the Swiss Federal Veterinary Office (FVO) in 1999. Whereas
some two million laboratory animals were still in use in 1983,
the figure had dropped to less than 500,000 in 1999. The precise
figure stated in the animal experiment statistics is 445 682
animals (1.5% less than the previous year).
Animal experiments requiring a permit are conducted primarily
in the areas of research, product safety, diagnostics and
teaching. Over 90% of experiments are directly related to
research concerned with human diseases. Around 70% of the
animals were used in industry and a quarter in institutes
and hospitals. The use of animals in basic research showed
a slight increase. In Switzerland, no animal experiments for
cosmetics or household products have been conducted since
1995. Nine out of ten laboratory animals are mice, rats, hamsters,
guinea-pigs or other rodents. Genetically modified mice, 6.5%
more overall compared to 1998, have also been used. In isolated
cases, the experiments have involved the use of fish, amphibians,
rabbits, monkeys, various domestic animal species and poultry.
About 7% of animals were subjected to serious harm in lethal
trials or trials involving a disease model with severe symptoms.
In most cases, these trials were designed to check the quality
of vaccines or develop and test new medicines. 25% of the
animals were subjected to moderate discomfort and 68% to little
or no discomfort. There were 2142 valid cantonal animal experiment
permits issued in Switzerland in 1999. 624 permits were newly
issued, over a quarter of these with attached conditions,
and 8 applications were rejected. As the monitoring authority,
the FVO requested more specific information or issued criticisms
in respect of 38 permits. 142,151 animals were used in animal
experiments not requiring permits. In most cases, these involved
the killing of animals in order to take organ or tissue samples,
or feed trials in large poultry herds.
4. Animals must be kept under humane conditions
The Swiss Animal Protection Act (TSchG) and Animal
Protection Ordinance (Tierschutzverordnung: TSchV) regulate
the protection, husbandry, care and handling of animals. A
complete shift in thinking about laboratory animal husbandry
occurred in the eighties. Previously, the emphasis had been
on standardising the keeping of individual animals under hygienic
conditions with optimised climate control and lighting, and
sterilised, standard feeds. But now the natural needs of laboratory
animals are increasingly taken into account. Hard, perforated
floors with no bedding to lie on are no longer deemed acceptable.
In addition to the need for group-based husbandry for the
higher mammals such as cats, dogs, pigs and monkeys, all laboratory
animals now have to be provided with facilities for playing,
climbing, hiding and resting.
If the experimental conditions require dogs to be kept separately,
for example in the measurement of metabolic rates using labelled
substances, the animals should at least remain in contact
with members of the same species, via smell and acoustically
and visually. Adaptation and training periods have also been
introduced for the animals. Other regulations introduced in
recent years also need to be taken into account: In the European
Convention for the Protection of Vertebrate Animals used for
Experimental and other Scientific Purposes (Council of Europe
Convention, 18 March 1986), crucial importance is attached
to the training of individuals who care for laboratory
animals and who conduct or arrange experiments. Good training,
and thus a good knowledge of the needs and rights of animals,
and their appropriate and technically correct use in experiments
can make a significant contribution towards avoiding extreme
discomfort.
5. All animal experiments are registered with the relevant
cantonal authority
Appropriate forms issued by the Federal Veterinary Office
must be completed in order to apply for permits and register
animal experiments. At Roche, all the relevant documents are
entered in the central Animal Registration, Management
and Accounting System (or TIGER – an acronym derived from
the German TierGesuchs- und Registriersystem), a system developed
jointly with sister companies in the chemical/pharmaceutical
industry, the Cantonal Veterinary Office and the Federal Veterinary
Office. These days, the Animal Protection Officer in
a company such as Roche or an institute is not just responsible
for the administrative processing of applications, permits
and surveys of experimental animal numbers. Working in conjunction
with the investigators and the laboratory personnel, he also
oversees the basic and advanced training of animal experts.
An animal experiment application must reveal whether the
animals derive from a recognised animal breeder, can be transferred
from an earlier trial or originate from some other source.
Details concerning the investigators' university qualifications,
specialist subjects and basic and advanced training are also
recorded.
The trials must be described in detail, including the objective
and the specific, direct question to be answered by the trial
and the basic knowledge obtained compared to other experimental
methods. Finally, the importance of the expected information
yielded must be evaluated and the status of the research described.
The trial design, including the method, plan and procedure,
preparation of the animals, including anaesthesia and pain
control, must also be stated on the application form. Explanations
concerning the methodology and interventions, operations,
substance administration and sampling in the animals in each
trial, the duration of the trial and the nature of the evaluation
all play an important role. Reaction tests, the recording
of pathophysiological parameters and statistical analyses
are also described.
Details must also be provided about the expected effects
on the animals' well-being (feed consumption, growth), severity
of side effects, husbandry and recovery, weighed against the
pain, suffering and anxiety experienced by the animals. The
reasoning must be stated for the stopping criteria, method
of killing, special features and advantages of the trial method
and the selection of the scheduled animal species and number
of animals.
If it is possible to take account of existing findings for
chemicals with a similar structure, the necessary animal experiments
can often be adapted to answer specific questions as a result
of such structure-effect correlations. In many cases, the
substance quantities to be administered can then be determined
in advance, thus dispensing with the need for preliminary
animal trials for dose selection.
Frog as donor of oocytes
Frogs (Xenopus laevis) serve as donor of oocytes for different
in vitro- experiments
Source: Daniela Jenni, PRBM-A, Roche
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