Alternatives to animal studies in Pharmaceutical research has been explained on the basis of replacement, reduction and refinement. Also newer pre-clinical animal models like use of genetically modified animals were presented.

1. Shri. Prashant Shivgunde

2.  What are laboratory animals used for?
◦ Animals are used in research and testing because
they are similar to humans, and because they can
be subjected to procedures which would not be
permitted with humans.

3. 1. To improve our basic knowledge of
complicated biological systems and effects
of diseases on these systems.
2. To develop new and better ways of
diagnosing diseases such as cancer and
heart disease.
3. To develop new surgical and medical
treatments for human diseases (e.g.
vaccines).

4. 4. To produce and test biological products that can
be used in the prevention and treatment of
diseases (e.g. vaccines).
5. To ensure that all medicines are safe and
effective.
6. To test chemicals to discover whether they are
likely to be harmful to human health. If a
chemical is found to be hazardous it might be
banned altogether, or its sale and use restricted.
This toxicity testing provides the information
which leads to hazard warning labels on many
products.

5. As a result of animal experimentation, both human
and animal suffering has been reduced. Some
examples of medical advances through animal
research are the development of:
1. MEDICINES, e.g. antibiotics and analgesics, and
medicines for treating asthma and diabetes;
2. VACCINES, e.g. for polio, measles and whooping
cough;
3. SURGICAL TECHNIQUES, e.g. heart surgery and
organ transplantation;
4. MEDICAL TECHNOLOGY, e.g. life-support
machines for keeping premature babies alive.

6.  The costs to the animals involved in scientific
procedures can be substantial.
 They can involve suffering caused by the
conditions under which the animals are kept,
 As well as by the scientific procedures to
which they are subjected.

7.  In the India, the Prevention of Cruelty to Animals
Act, 1960 and CPCSEA place controls upon the
use of protected animals in experiments.
 Non-animal methods must be used whenever
possible and animal suffering must be
minimised.
 The Act requires that the project, the researcher,
and the premises all have licences.

8.  Thousands of organisations throughout the
world are concerned with the issues of animal
experimentation and vivisection.
PETA
BUAV
NAVS
FRAME
AMRIC

11.  The issue of animal experimentation is very
complicated. For example, the more animals are
like humans (e.g. monkeys),
 Because of differences between species, will
never provide results which are perfectly relevant
to man.
 However, they do often reveal the unexpected.
Non-animal methods tend to tell us expected
things, but are less likely to reveal the complex
events which occur in a living animal or human.

12.  The animal welfare case against animal
experimentation is very strong.
 However, another convincing argument is
that it would not be proper to deny the
benefits that animal experimentation can
offer, both to science and to the sick and
suffering, until suitable alternatives for
animal procedures have been found.

13.  Before experimentation, CONSIDER following
alternative methods -
◦ Use of The 3 R’s
◦ Use of High Throughput screening
◦ Use of genetically modified (GM) animals
◦ Use of human cells and tissues for research and
testing
◦ Use of primates in research
◦ Risk assessment in the safety testing of chemicals
and products
◦ Vaccines and animal testing

15.  Replacement alternatives can be defined as
methods or strategies which do not involve
the use of protected animals in regulated
procedures.
 In the UK, animals can only be used if no
replacement alternative is available. Since the
Animals (Scientific Procedures) Act 1986
came into force, a number of alternatives
have been developed

16.  Pyrogens are substances that, if introduced into the
bloodstream, cause a dramatic increase in body
temperature.
 The most common pyrogens are substances which are
parts of the cell walls of bacteria.
 Even when the bacteria are killed by sterilisation, these
substances remain.
 Even a tiny quantity of these pyrogens can cause a
lifethreatening reaction if injected into a patient.
 Any drug that is given by injection must be guaranteed not
to contain any pyrogens.

17.  A sterile solution is injected into the ear veins of
rabbits.
 The rabbits’ body temperature is then measured
for several hours.
 This test takes a long time, requires training and
experience to perform accurately, and is
expensive.
 Hundreds of thousands of rabbits are still used,
world-wide, for this test each year.

18.  This was developed as an alternative to the rabbit
test.
 Blood taken from horseshoe crabs (Limulus
polyphemus) is mixed with the substance being
tested in a test tube.
 If bacterial pyrogens are present, the blood clots.
 Roughly a third of the blood of the horseshoe
crab is taken and then it is released alive.
 Nevertheless, roughly 30,000 horseshoe crabs
die each year as a result of providing their blood.
 This test is much more sensitive than the rabbit
test.

19.  This test does not require any animals to be
used.
 It uses human blood (from volunteers), which is
mixed with the test substance in a test tube.
 Any bacterial pyrogens present cause one type of
blood cell (monocytes) to release cytokines.
 This is what occurs naturally inside living animals
and causes the increase in body temperature.
 The amount of cytokine produced depends on
how much pyrogen was present in the test
sample, which can be measured by a highly
sensitive immunoassay:

20. • It is cheaper, faster and more sensitive than
the rabbit test.
• It predicts the response of human patients
more accurately than either the rabbit test or
the LAL assay.
• It does not cause suffering of any animals.

21.  Monoclonal antibodies (mAb) have proved to
be important in scientific research, clinical
diagnosis and therapy.
 An increasing number of drugs are based on
the ability of mAbs to ‘home in’ on particular
sites within the body.
 mAbs are generally produced by first
injecting a number of mice with the
substance of interest.

22.  The animals produce antibodies against the
‘foreign’ substance as a natural immune
response.
 Selected animals are killed, their spleens are
removed, and the antibody-producing cells are
fused with a cell line that can grow continuously
in culture.
 The resulting fused cells (hybridomas) are then
cloned to select individual cells secreting the
antibodies of particular interest.
 The selected hybridomas then have to be grown
up to produce sufficient antibody for purification
before use.

23.  This can be done either in vitro or in vivo.
 The in vivo method involves injecting a mineral
oil, which acts as an irritant, into the abdominal
cavities of mice or rats.
 Roughly a week later, the hybridoma cells are
injected, which multiply and secrete the
monoclonal antibody into a fluid (ascites fluid)
which accumulates in the abdomen.
 The increasing volume of fluid swells the
abdomen, causing severe discomfort, before it is
collected through a hypodermic needle and the
animal is humanely killed.

24.  The alternative in vitro method involves the
growth of hybridoma cells in large volumes of
culture medium and the purification of the
antibody from this culture medium.
 Initially, the main disadvantages of this method
were that the antibody was present in much
lower concentration in the culture medium than
in the ascites fluid, and that some hybridoma
cells would not grow well in cell culture.
 These difficulties have been mostly overcome as
a result of advances in cell culture techniques.

25.  Since cell culture techniques have improved,
they are now a satisfactory alternative to
using animals for the production of
monoclonal antibodies.
 In Europe, the ascites method of production
is no longer permitted.
 In this case, the adoption of a non-animal
alternative has saved the suffering of many
thousands of animals.

27.  Phototoxicity is a skin reaction which occurs
following exposure to certain chemicals and
subsequent exposure to sunlight.
 In some cases this can lead to severe
inflammation and blistering.
 It is important to test, for phototoxicity, all
the components of products that will be
applied to the skin.

29.  The standard animal test for phototoxicity uses
guinea pigs that have had a patch of skin shaved
and the test chemical applied.
 Half of the guinea-pigs are then exposed to
ultraviolet light for two hours or more.
 The animals are observed for several days to
check for inflammation and swelling.
 The animals must be restrained throughout the
whole procedure, and painkillers cannot be given
as they might lessen the inflammation.

30.  An alternative method has been developed that
does not require the use of live animals.
 The test chemicals are applied to mouse skin cells
grown in cell culture.
 One set of cell cultures are exposed to a known
intensity of ultraviolet light,
 while duplicate cultures are kept in the dark.
 A dye called Neutral Red is then added to both sets
of cells.
 If the cells are alive and well, they take up the red
dye; if the cells are dead or dying, they do not.

31.  The difference in dye uptake between the light-exposed
cells and those kept in the dark gives a measure of the
phototoxicity of the test substance.
 This alternative test has been proven useful for
predicting phototoxicity in humans
 This Replacement test, relying on the response of
cultured cells in vitro, is now the recommended test for
chemical photoxicity.
 Apart from not causing any animals to suffer, it has the
advantages of being relatively rapid and inexpensive.

32.  In some laboratories, animals are still used, even
though the Organisation for Economic Co-operation
and Development (OECD), in its published test
guidelines (TG) for the testing of chemicals, has
approved
◦ Corrositex® (OECD TG 435),
◦ EpiDermTM and
◦ EPISKINTM (OECD TG 431)
for testing skin and eye irritation and corrosivity.
 OECD TG 432 gave approval to the in vitro 3T3 NRU
phototoxicity test, and OECD TG 428 gave approval
to in vitro dermal absorption methods.
 All these tests have been validated by the European
Centre for the Validation of Alternative Methods
(ECVAM).

33.  In November 2008, the Interagency
Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) published
recommendations on pyrogenicity testing,
noting that while none of the five in vitro
pyrogen test methods evaluated “can be
considered as a complete replacement for the
rabbit pyrogen test (RPT) for all testing
situations …. However, … they can be
considered for use on a case-by-case basis
….”

34.  In this presentation, you have seen about
three Replacement alternatives to older tests
that were based on the use of animals.
 These new methods are examples of the
advances that can be made when scientists
actively work to find alternatives to laboratory
animal use.

35.  Animals are used in experiments when
studies in humans would
◦ not be permissible, when suitable non-animal
methods are not available and
◦ when they exhibit similar responses to people.

36.  An appropriate experimental strategy ensures
that the minimum number of animals is used.
 However, if too few animals are used,
experiments may have to be repeated, so a
balance must be struck between achieving
reduction and ensuring that the aims of an
experiment are met.

37.  Reduction can be achieved through clearly
defined aims, good experimental design and
appropriate statistical analysis.
 Poorly designed and badly analysed
experiments, which use animals inefficiently,
are unethical and are scientifically
unsatisfactory.

38.  To assess whether a new medicine (A) could
reduce the size of tumours more effectively
than treatment with existing medicine B,
researchers investigate the response of
groups of animals to each medicine.
 Statistics are used to assess the probability
that any differences which are found
following the treatments are not due to
chance variation, but may be due to a
genuine effect of the treatment.

39.  Small tumours are transplanted under the skin of
a group of mice.
 Individuals are then assigned at random to
treatment with medicine A or medicine B, or non-
treated controls, and the sizes of the tumours are
measured over a period of a few days or weeks
depending on the type of tumour.
 The mean (average) tumour diameter for each
group is measured to test the hypothesis that
medicine A was more effective than medicine B in
preventing tumour growth.

40.  Individual mice in a group will vary in their
response (e.g. to a medicine).
 In addition to this biological variation there
could be errors in making measurements and
observations.
 Good experimental design and practice both
minimises and takes account of these
variations, so that fewer animals are required
for a study and valid results are obtained.

41.  In a well-designed study, with adequate
statistical power, researchers may detect a
difference between the two groups of
animals, which statistical analysis suggests is
unlikely to be due to chance.
 In given example, this could indicate that
medicine A might be a promising new
treatment for cancer patients and should be
studied further.

42.  Controlling experimental variables can produce more
reliable data and, therefore, reduce animal use.
 To be confident that the responses in the medicine A
treatment group were due to the medicine, the researcher
must be sure that other factors were not responsible for
the results obtained, such as diet, group size, noise levels,
humidity and lighting.
 The researcher must ensure that only the variables under
study (e.g. medicine treatment) differ between the two
groups and that all other variables are controlled.
 For instance variation in temperature and levels of stress
can alter an animal’s response to a medicine, leading to an
invalid conclusion.
 Techniques that minimise the stress experienced by
animals can also minimise variation and reduce animal
use.

43.  Specific recommendations for environmental
standards exist in many countries.
 Many studies follow the requirements of
Good Laboratory Practice (GLP),
◦ a set of guidelines that detail how a study should
be conducted, including details of animal
husbandry and diet, and which set a standard for
quality assurance.

44.  One of the key methods of achieving reduction is
to minimise variation by using animals that are
as similar as possible.
 For example out bred mice can differ greatly in
size and body weight, and in their responses to
medicines.
 In contrast, animals of inbred strains are very like
identical twins, and tend to exhibit similar
responses.
 Researchers can achieve reduction by minimising
genetic variability through the use of inbred
strains.

45.  Health status and high standards of care
must be ensured.
 Unhealthy or stressed animals may respond
differently to a medicine, increasing variation
in the experimental results, so that the
number of animals used may need to be
increased.
 For instance, in given example, the animals
used should be disease free, apart from the
implanted cancer which was being studied.

46.  Regrettably, many experiments are poorly
designed, resulting in unnecessary animal use.
 During the analysis of experimental data the
wrong statistical test is sometimes used, so that
the conclusions from the results are invalid.
 So it is strongly recommend that a statistician is
consulted at the initial planning stage to ensure
that the study is correctly designed and analysed,
and that an appropriate number of animals is
used.

47.  If it is impossible to gain the necessary scientific
information without using live animals.
 And suppose that every effort has been made to
reduce the number of animals to as few as possible.
 It then becomes necessary to use refinement
techniques to reduce the amount of pain and distress
caused to the animals to an absolute minimum.
 Refinement techniques can have a great effect on
improving the well-being of laboratory animals, as
well as enhancing the value of the experimental data.

48.  Laboratory animals spend most of their time in cages
or other enclosures, rather than actually undergoing
experimental procedures.
 If animals are kept in cramped and unsuitable
conditions, where they cannot behave as they would
normally, they show signs of stress.
 Animals all need certain things to improve their
environment.
 It is important to consider the particular needs of
each species, when designing their housing and when
planning to introduce environmental enrichments.

49.  Rats and mice are the animals most commonly used in
medical experiments and for safety testing.
 It is therefore very important that every effort be made to
improve their living conditions.
◦ Rats and mice are naturally social animals, and should normally be
housed in groups.
◦ They will explore their surroundings, and will build nests and find
somewhere to hide from danger. Simply adding shredded paper
and pieces of wood to their cages will allow the animals to behave
more normally.
◦ A little time spent gently handling young rats and mice makes
them less shy and much easier to handle when they are adults.
 If the animals are not afraid of being handled, any future
experimental procedures will be less stressful for them.

50.  Dogs are still used for the safety testing of some new
drugs.
◦ They should be housed in small groups.
◦ Dogs will naturally roam their environment. They should be
given as much room as possible, and should be given
suitable objects to chew or play with.
◦ They should be given somewhere to seek shelter, and an
elevated platform to act as an observatory.
◦ Dogs will benefit from human attention.
 Allowing the staff that care for laboratory animals
time to spend grooming the dogs will reduce stress
in the dogs and allow them to be handled with
greater ease.

51.  Fish, especially zebrafish, are being widely
used for research.
 It has been shown that providing aquatic
plants (even if they are artificial) results in
more-natural behaviour and better breeding
success.

52.  Like humans, animals have the capacity to feel pain.
 It is therefore important that great care be taken to
design experiments so that they are likely to cause as
little pain and distress as possible.
 Pain can cause changes in animals that are difficult to
predict and that can make the scientific study
worthless.
 This means that effective anaesthesia for surgery and
effective pain relief, not only benefit the animals, but
are essential for providing meaningful results.

53.  Anaesthetics are used to prevent animals sensing
pain during any necessary surgery or other procedure
that would be painful.
 An ideal anaesthetic would put the animal into a
rapid ‘deep sleep’, so that it could not feel any pain
or discomfort throughout the surgical procedure, and
would allow the animal to ‘come round’ soon after
the operation was complete.
 It is important to choose the correct anaesthetic for
each species.
◦ For example, the anaesthetic, isoflurane, is very effective in
rats, but causes distress in rabbits.

54.  This is a question that must be asked if we are to treat
animals with pain killers effectively.
 Anyone who has kept a dog, or a cat, as a pet will feel sure
they can recognise when their pet is in pain.
 However, it becomes more difficult with species such as
rats and mice to find out without experts. As they are a
prey species in the wild, they have evolved so that they do
not show signs of pain and distress in their behaviour.
 One way that the well-being of rodents can be assessed is
by monitoring their weight.
 If an individual’s weight drops, it is a sure sign that it is
suffering in one way or another.

55.  Scientists look for the earliest possible signs
providing information they require.
 As soon as sufficient evidence is obtained, the
experiment should be ended (the endpoint).
 In most cases, the best course of action is then to kill
the animals humanely, without causing further pain
or distress.
 Recently, a better understanding of the underlying
biology, or the development of new technologies, has
allowed sufficient information to be gathered at an
early stage, before the animals are in severe pain or
distress.

56.  The testing of anti-cancer drugs often involves giving
drugs to mice that have growing tumours.
 The effect of the drug is judged by how quickly the tumour
increases in size.
 In the past, for this to be measured accurately, the tumour
would be allowed to grow very large, sometimes weighing
as much as the body of the mouse.
 But nowadays, highly sensitive imaging can be used to
monitor tiny tumours long before they cause significant
pain or distress to the animals.
 This can be achieved by scientists placing genes into the
cancer cells which makes them give out light.

57.  Animal tests are used to identify chemicals that cause
allergic skin reactions.
 For a long time, the accepted animal test caused
them to suffer similar, painful, skin reactions.
 However, a better understanding of the underlying
biology of allergic reactions has now allowed the
development of a new test.
 This new test uses mice — but can accurately
measure the risk that a chemical will be hazardous,
without the animals having to suffer the type of
painful reaction that we seek to avoid in humans.

58.  At present, there is no accepted method to
predict the hazard of allergic skin reaction
that does not use animals.
 However, there is a great deal of research
aimed at developing such a method by using
sophisticated cell culture models and
computer-based predictions based on
chemical structures.

59.  Refinement is important for both animal welfare
and scientific reasons.
 Improving animal welfare through refinement
techniques is not at odds with the need to
produce good science.
 In fact, if animals are free from stress, and any
unavoidable pain is reduced to a minimum, it is
much more likely that the experimental
procedures will produce reliable results.

61.  To understand the complexity and functioning of
human body systems and their control
mechanisms.
 To get information which is directly relevant to
human diseases and their treatment, where
studies on human volunteers would not be
permissible.
 Moreover,
◦ It can avoid the problem of species differences,
◦ In addition, human cells can also be useful in testing the
safety and efficacy of many types of chemicals and
products

62.  The kinds of organs and tissues that can be
obtained for use in research include the
◦ skin,
◦ eyes,
◦ liver,
◦ placenta,
◦ lungs and kidneys
◦ as well as heart valves, blood vessels and blood
cells

63.  Human cells and tissues are normally obtained
directly from donors in four main ways:
◦ (a) from patients undergoing routine surgical operations;
◦ (b) from patients who die in Intensive Care Units;
◦ (c) from patients who die in Accident and Emergency
Units;
◦ (d) during post-mortem examinations.
 The use of organs and tissues for transplantation
must have the highest priority, but
 where they are unsuitable for this purpose, their
use for research can be considered.

64.  Patients undergoing routine operations, including
plastic surgery, can elect to donate tissue such as
skin, for research projects.
 New mothers can also donate their placentas after
they give birth.
 Patients who die in Intensive Care Units are classified
as potential heart-beating donors. In such cases,
tissues can, with permission of the next-of-kin, be
taken for research purposes when the patient is
declared to have undergone brain stem death.
 It is also possible to retrieve full-thickness skin
tissue, usually from the abdomen, up to three days
after death, during postmortem examinations.

65.  The amount and variety of human tissue that can be
made available for research purposes is greatly
limited by legal, ethical, cultural and practical factors.
 There is a formal system in the UK for obtaining
human tissue for medical purposes, that involves
transplant co-ordinators and couriers who work in
conjunction with surgical staff at hospitals.
 Transplant co-ordinators can often assist in the
supply of tissues and organs that are unsuitable for
transplantation or are surplus to other medical
requirements.

66.  In the past, human cells and tissues for research and
testing have often been obtained in an informal way.
 This has restricted the general availability of such
material, and has caused problems, particularly when
donors or relatives were not asked for their informed
consent in an acceptable way.
 Obtaining informed consent involves providing a
written statement of how and why the tissues and
cells would be used, disclosing any potential risks to
the donor (or the relatives), followed by an
opportunity for further discussion, so that the donor
(or the relatives) can freely make a personal and
educated decision.

67.  It is advantageous to have a formal procedure for
acquiring, processing and distributing human tissue.
 To take account of important ethical, legal and scientific
requirements, human research tissue banks are being
established in several countries.
 Tissue banks can establish call centres, so that tissue can
be collected rapidly and safely.
 Human tissue banks are staffed by people who can
process the tissues according to the specific requirements
of individual researchers, and can maintain stores of tissue
to be supplied according to demand.

68.  The tissue can be monitored for disease status
and other properties.
 Also, tissue banks can demand that researchers
have obtained ethical approval for their work.
 All human cells and tissues for research and
testing should be accounted for by logging
tissues onto a computerised database, in order to
monitor their use and distribution.
 Donor confidentiality can be maintained by using
an anonymous tracking system

69.  There are potential safety problems due to
possible contamination of tissues with
disease-causing viruses, such as HIV and
hepatitis.
 It is not possible to screen living patients for
these diseases without their consent.

70.  Human tissues can be used in many different
ways, ranging from fundamental studies
conducted to gain an understanding of the
structure and function of human cells in health
and disease to efficacy and safety testing.
 Both types of studies can also involve
experiments to see how substances might be
altered chemically in the body, as a result of
enzyme action (metabolism), especially by using
human liver cell models.

71.  Also, human cells can be used to model the
absorption of chemicals and their passage across
and through various barriers in the body, such as
◦ the intestinal wall,
◦ the blood-brain barrier (passage into the brain) and
◦ the skin (e.g. with human skin models).
 In this way, many cosmetics products and their
ingredients are now being assessed for toxicity,
such as skin irritation.

72.  In safety and efficacy testing, once the first
studies with human cells in culture have been
completed, subsequent tests might involve
animals and also volunteers and patients.
 However, these later investigations are often
conducted only on those chemicals that have
performed satisfactorily (i.e. that have the
desired therapeutic activity without toxicity)
in the earlier tests.

73.  Studies on human tissues are needed as
alternatives to using human volunteers, as
well as to replace the use of animals.
 This is because more needs to be known
about how a new chemical or medical
treatment will affect humans before clinical
trials in humans are conducted.
 Such studies can reduce the chances of
unexpected and life threatening toxicity.

74.  The use of human cells and tissues can
provide the basis for the development of very
useful and predictive methods for replacing a
wide range of animal experiments.
 In particular, the use of human cells and
tissues can be more beneficial than using
animals by providing more-relevant
information.

75.  However, the main constraint on using human
cells and tissues is their safe, reliable, efficient
and ethical supply to researchers.
 The advent of human research tissue banks
should help to meet these needs, but it is crucial
that volunteers, patients and relatives of
deceased individuals are encouraged to donate
tissues, following receipt of the necessary
information and reassurances and are convinced
that the proper procedures will always be
followed.

76.  Information that determines bodies look and function
stored in nuclei of the cells, in the form of GENES.
 Changes in specific genes or the number or structure of
chromosomes can also lead to specific diseases
 Change can be done by- inserting, mutating or deleting
genes
 This can help to produce models of human disease in
animals, in order to develop treatments for them.

77.  To increase knowledge of normal human development and of
the functions of the body’s organs and systems
 To study diseases and in the development of new medical
treatments
 To produce useful biological products,
 For the safety testing of vaccines and chemicals,
 For animal to human organ transplantation research,
 To increase the production and the quality of products from
farm animals.

78.  GM animals show- how genes are regulated and how
they affect the normal development and functions of the
body
 E.g.- Introducing transgenes that alter the amount of
functional insulin-like growth factor that is expressed in
the body

79.  Genes role in
◦ to the development of disease,
◦ to mimic human diseases,
◦ to investigate new treatments for diseases.
 E.g. of GM models exist for a variety of human diseases,
◦ cystic fibrosis, rheumatoid arthritis and Alzheimer’s
disease.
 However, these GM animals might not necessarily be relevant
models of these diseases in humans.

80.  Biological products can be used to treat certain human
diseases, but such products are often expensive to
make.
 For example, large amounts of human protein (alpha-1-
antitrypsin) are used to treat a life-threatening
condition called emphysema.
 GM sheep have been developed, which make the protein
in their milk in larger quantities than could be produced
by using cell culture methods.

81.  GM animals
which produce
useful biological
products can be
created by the
introduction of
the DNA which
codes for a
particular
product.

82.  GM mice are being developed for use in testing the
safety of vaccines before they are given to humans.
 For example, GM mice can now be used to test the
safety of batches of poliovirus vaccines, and this should
replace the current practice of using monkeys for this
purpose.

84.  To perform toxicity or safety testing.
 GM animals have been produced that carry
genes that make them more sensitive to toxic
substances.
 This allows results to be obtained more
quickly, with fewer animals, and with less
animal suffering

85.  To provide organs for patients who require a
transplant- Pigs containing human genes in their cells
have been produced
 Under normal circumstances, an organ transplant from
an animal would be rejected by a patient’s immune
system
 Inserting a human gene which causes a human protein
present on the surface of the animal cells to hide them
from detection by the human immune system, it is
hoped that the organ would be recognised as “human”
or “self”

86.  One problem with this new technology is that adding
one human gene to the cells of the animal is not
enough to stop the activities of other components of
the very complex human immune system, which could
still reject the organ.
 There is also concern that such operations could lead to
the spread of diseases from animals to humans.

89.  Many people object to genetic modification,
because the production of GM animals can
seriously affect animal welfare in several ways
 some of the methods for producing GM animals
give unpredictable results
◦ GM animals suffering from deformities, diseases and organ
failure-
 genes being inserted into the wrong place within a
chromosome,
 deleting a gene can have unpredictable effects on other
genes,
 foreign gene cannot be handled correctly by the animal

90.  Only a small percentage carry the desired
foreign DNA, and not all of these will survive.
 Some surviving animals that do not express
the foreign gene might be used for other
purposes, but many others will be killed.
 Disease models designed to exhibit
corresponding human diseases, are likely to
suffer uncontrollable pain and distress.

91.  Is it morally acceptable
for humans, for their own
purposes, to manipulate
the fundamental genetic
make-up and principle
characteristics of other
animals in this way?

92.  There are natural, fundamental genetic differences
that separate species from one another.
 GM technology has the potential to remove some of
these differences by transferring genes between
widely differing species.
 An example is the transfer of human genes into other
animal species, which could therefore produce
animals that are more human.
 To modify animals drastically by using GM technology
could radically change the direction of evolution.

93.  Some studies of human diseases are now
possible by using genetically modified
invertebrates, such as fruit flies.
 For instance, it has recently been
discovered that these flies have cells that
function like the cells of the human
pancreas and so might be used instead of
GM mice to study human diabetes.

95.  The other possibility is to use GM human or
animal cells to express human proteins.
 This is one way of producing large amounts
of biological products such as alpha-1-
antitrypsin without using sheep.
 Similarly, yeast and bacterial cells can be
used instead of GM animals for some types of
toxicity testing.

96.  GM animals can be useful for studying diseases,
developing medicines and for toxicity testing.
 However, the number of scientific experiments involving
GM animals has increased dramatically over the last few
years and it is likely that these numbers will continue to
increase.
 This is worrying, since many GM animals may suffer or
even die as a result of having their genetic make-up
altered, without providing useful information about human
diseases.
 It is important that GM animals are used only as a last
resort, and their use is subject to strict control in view of
their potential suffering.

97.  By considering the welfare and likely needs of
each GM animal, encouraging the sharing of
stocks of animals, gametes and embryos, and
carefully designing all experiments in which
they are employed,
 it should be possible to avoid the use of such
large numbers of animals and to minimise
their suffering, where such use cannot be
avoided altogether.

98.  Embryos of useful
GM strains could be
stored frozen in
liquid nitrogen.

100.  Can we produce vaccines without using
animals?
 Can viruses only multiply inside a host cell?
 Can we use any other method?

101.  For example, microorganisms can be
◦ Grown in large bioreactors and
◦ killed by using chemicals, heat treatment or gamma
irradiation.
 Viruses can only multiply inside a host cell,
◦ So they are grown in cell cultures (fertilised chicken eggs or
in living animals)
 Today, only very small numbers of animals are
used for this purpose.
 It has been possible to replace cultures made from
fresh tissues (require animals to be killed) with
cultures of permanent cell lines.

102.  During the production of a vaccine, it is necessary to
carry out safety tests and potency tests.
 The way that these tests are carried out is laid down in
regulations made by the international agencies.
 Unfortunately, many of these tests still require living
animals.
 Safety testing involves mice, guinea-pigs and monkeys
being injected with each batch of product, to see if they
become ill as a result of harmful impurities.

103.  Potency tests also involve injecting animals with the
product and later exposing them to the infectious
microorganism, to compare the infection rate with that
of animals that were not vaccinated (challenge test).
 These tests, especially the potency tests, often cause
severe distress and suffering to the animals.

104.  Due to reliable results, Potency tests for diphtheria and
tetanus toxoid vaccines have been modified to allow the
use of fewer animals
 Potency tests for a number of other vaccines do not
require the use of any animals,
◦ since it is possible to directly measure the number of live bacteria
in culture medium.
 However, potency tests for inactivated and toxoid
vaccines require animals in most cases.
 Use of GMP and sophisticated methods of analysis
reduced impurities level

105.  Since January 1997, the European Pharmacopoeia, which
lists most tests that need to be done on medical
products, has not required the safety testing of many
products, including diphtheria, tetanus and pertussis
vaccines.
 This decision will probably be extended to other
vaccines in the near future.
 The Three Rs concept

106.  NO challenge testing-
◦ Attempts are being made to refine potency tests by measuring the
antibodies produced in the serum of the animals after they have
been injected with the vaccines, rather than by doing a challenge
test involving the injection of the infectious organism.
 Application of ELISA-
◦ Methods such as the Enzyme-Linked ImmunoSorbent Assay
(ELISA) can be used to measure these serum antibodies very
accurately.

107.  Developing vaccine not containing organism-
◦ Developing new vaccines which do not contain the organism that
causes the disease which can be tested them successfully by using
non-animal methods.
 To produce these vaccines without using animals-
◦ In some cases, it may also be possible to produce these vaccines
without using animals or animal cells at all.
 For example,
◦ Plants are being infected with genetically-engineered plant
viruses, to make the plants produce proteins which may possibly
be useful as vaccines against malaria, hepatitis B and foot-and-
mouth disease.

108.  Hope-
◦ These plant-derived products will not need to be purified or
refrigerated.
 Attempts are being made to produce genetically-
engineered vaccines against HIV and hepatitis.
◦ Antigens which cause an infected individual to produce an
immune response are coded for by different genes in the
microorganism.
◦ Once a gene coding for an important antigen is known, it can
be put into a harmless virus or bacterium of a completely
different sort. This organism can then be used to produce a
safer vaccine.

109.  Guidelines encourage the use of non-animal
methods wherever possible.
 Animal use in vaccine production can already be
reduced by following the principles of the Three
R’s.
 As we find out more about the immune system
and about how vaccines work, we will be able to
develop better non-animal methods for
producing and testing vaccines that are safe and
effective.

110.  Risk - probability that something (like a
chemical) will be harmful,
 Risk assessment- process whereby this
probability is estimated.
 Exposure to chemicals is unavoidable, since
they occur everywhere

113.  Hazard is the potential to cause harm.
 Hazard depends on chemicals physical and chemical
properties, and the effects that it has on the
organisms with which it comes into contact.
 Often, hazard potential can only be determined by
toxicity testing.
 Non-animal methods for toxicity testing are being
increasingly used, including computer modelling, cell
and tissue culture (in vitro testing), and organisms
such as bacteria and fungi

114.  Toxicity testing does not exactly represent
what occurs in real life
◦ Species differences
 Not a good model for predicting the effects
of a chemical on large populations of humans
over a lifetime of exposure to very low doses
 Different individuals of the same species can
also react differently

116.  Efforts to refine the use of uncertainty factors are
ongoing
 in vitro methods – to avoid species differences
◦ E.g. human cells rather than animal cells or living
animals.
 Problem - the effects on a single type of cell may
not correspond to its effects on whole organs or
a whole living animal or human.
 Use of variety of human cells in tissue equivalent
models, such as reconstituted skin and eye
models.
 Eventually, use of tissue equivalent models may
mean that animal testing is no longer necessary

118.  Sometimes, the type and
level of risk from a
chemical can be
considered acceptable, if
the benefits of using it are
thought to outweigh the
risks (risk–benefit
analysis).
 The risk of a medicine Vs
Cosmetics causing an
adverse effect, such as a
rash,

119.  Risk can also be managed by minimising exposure, for
example:
a) by using secure packaging for products containing
potentially hazardous ingredients;
b) by providing hazard warnings and labels;
c) by avoiding or limiting exposure of particularly
vulnerable groups, such as the young, the elderly, or
those with certain disease conditions;
d) by imposing a complete ban on the use of the
chemical; or
e) by substituting the chemical with a safer alternative.

120.  Major reason why toxicity tests continue to be
undertaken in traditional ways is the perception
among many toxicologists that dose–response data
from traditional animal test methods is essential for
undertaking quantitative risk assessments.
 However, many scientists are already looking for
better ways of conducting risk assessments, through
the development and use of new and scientifically
advanced non-animal methods and models.