nikhil joshi ntc

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Experiment No. Date:

NATIONAL TOXICOLOGY CENTRE VISIT

INTRODUCTION

NTC (National Toxicology Centre) , has been conducting acute, sub-acute toxicity testing sinceAugust 1993. With expertise in many areas of Drugs, Agrochemicals, Water treatmentChemicals, Pharma packaging materials, test for bio-degradation of plastics, Antibodies

production, Research and safety evaluations. They serve the testing needs for both industry andpersonal. Complete support services including consultation, protocol development, quality

assurance, analytical chemistry and archive facilities are integral components of studiesperformed at NTC. Their technical and support procedures are in full compliance with FDA,

ISO and CPCSEA.NTC is located in Pune, on Sinhagad Road, just 10 Km. away from Swargatebus station.

Accreditation

NTC (National Toxicology Centre) is registered with the following governmental agencies:y FDA Approved

y ISO 9001:2000 Certified By Moody International Certification Ltd.

y CPCSEA Registered

y IEC

Animal experimentation or animal research, in vivo testing, and vivisection have similar

denotations but different connotations. The word has a negative connotation, implying torture,suffering, and death. The word "vivisection" is preferred by those opposed to this research,

whereas scientists typically use the term "animal experimentation". Before many drugs andcosmetics are tested on humans or released for human consumption, they are usually tested on

animals. This is such a common occurrence that poison doses are measured in LD50 medianlethal dose required to kill half the members of a tested population (usually rats) this involves

intentionally killing large numbers of animals. Many people are opposed to testing on animalswhile many others are for it.

NECESSITY FOR ANIMAL TESTING

Human biology is very much like that of many other animals. That is why, results from animal

experiments apply to people. Most laboratory animals have the same set of organs - heart, lungs,liver, and so on which work in the same way as they do in human. There are five major areas of

medicine and biology where animals for experiments need to be used.

Fundamental biological and medical research: Fundamental research in biology andmedicine are basic foundations on which future discoveries are based.Developing new treatments for diseases - There are many diseases which are yet to

have a proper cure like multiple sclerosis, certain cancers, as well as new diseases like AIDS,Alzheimer disease etc. All these need initial input in terms of animal experiments.

Preparations of natural products used in medical research and treatment -Animalscan produce useful medical substances in their blood or milk, like antibodies, vaccines and

hormones which are important for diagnostic tests, medical treatments, and basic research.


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Safety testing of chemicals and drugs - A wide range of chemicals and medicineswhich are used in day-to-day life, as household products, in farming, industry etc., need to be

tested for their safe use in humans as well as in animals.Study of genetic disorders - There are many diseases which are inherited fully or

partially and are caused by basic faults in a person's genetic code.

COMMITTEE FOR THE PURPOSE OF CONTROL AND SUPERVISION ON

EXPERIMENTS ON ANIMALS (CPCSEA)CPCSEA is the ethical committee responsible for all the control and supervision for the

experiments carried out on ANY animal before using it for experimentation. All animals must beacquired lawfully as per the CPCSEA guidelines. A health surveillance program for screening

incoming animals should be carried out to assess animal quality. Methods of transportationshould also be taken into account .Each consignment of animals should be inspected for

compliance with procurement specifications, and the animals should be quarantined andstabilized according to procedures appropriate for the species and circumstances.

The basic goal of these committee guidelines is to promote the humane care of animals used inbiomedical and behavioral research and testing with the basic objective of providing

specifications that will enhance animal wellbeing, quality in the pursuit of advancement ofbiological knowledge that is relevant to humans and animals. Some more points this committee

focuses on are:

y ANIMAL EXPERIMENTATION INVOLVING HAZARDOUS AGENTS

y MULTIPLE SURGICAL PROCEDURES ON SINGLE ANIMAL

y ANIMAL DURATIONS OF EXPERIMENTS

y PHYSICAL RESTRAINT

y PHYSICAL RELATIONSHIP OF ANIMAL FACILITIES TO LABORATORIES

Guidelines for Animal House Facilities

The animal house facility is available to boost the teaching, training and research facilities andto meet the growing demand for high quality laboratory animals in emergent field of

Experimental Pharmacology. Qualified veterinarians and technical personnel are always athand to help carry out research and keep it in accordance with the standards established by

the Committee for the Purpose of Control and Supervision of Experiments on Animals(CPCSEA COMMITTEE FOR THE PURPOSE OF CONTROL AND SUPERVISION ON

EXPERIMENTS ON ANIMALS (CPCSEA, Hon.Smt. Maneka Gandhi Drafted underChairperson, CPCSEA).

Animal care

The laboratory houses rats, mice and rabbits. Each species of animals is housed in barriermaintained individual rooms to avoid disease transmission and inter-species conflicts. Allefforts are made to maintain the animals under controlled environmental conditions

[Temperature (22-26C), RelativeHumidity (60 10 %), 12 hr alternate light and dark cycle]with 100 % fresh air exchange in animal rooms and uninterrupted power and water supply.

The support staffs ensures that every part of animal care including feeding, watering,restraining, cage cleaning, record keeping and ordering of animals, feeds, bedding materials

and equipment from outside sources are monitored. The animals themselves are isolated from


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human habitation and guarded from dust, smoke, noise, wild rodents, insects and birds-in factanything that would disturb their habitat. A high degree of hygienic conditions (macro- a-

environment) around the animals are maintained as per the CPCSEA guidelines.

Veterinary care

Periodic health monitoring of the animals is carried out to ascertain the heath status of theanimals. Veterinary care is crucial to keep the animals content and healthy. The veterinariankeeps them under daily observation and monitors any behavioural changes or signs of illness.

He supervises their diet and sanitation and contributes to the formulation of appropriate policiesand procedures for ancillary aspects of veterinary care.

Animal ethics

The Animal House is registered with the Committee for the Purpose of Control andSupervisionof Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Govt. of India

(Reg. No ) and has an established Institutional Animal Ethics Committee(IAEC) that advises thestudents, teachers/researchers on facilities, policies and practices concerning the care and use of

animals.

Ethical issues

Is it morally acceptable to cause pain, suffering and death to animals?

The question of defining the moral status of humans and animals often arises in the debate onresearch involving animals. Are humans morally more important than all animals? Is there a

sliding scale with humans at the top and the simplest animals at the bottom? Or are humans andanimals morally equal? We suggest that the proper moral treatment of a being depends on the

characteristics it possesses, rather than simply on the species to which it belongs. Mainly fivemorally relevant features:

y Sentience (the capacity to feel pleasure and pain)

y Higher cognitive capacities (for example, the ability to use language and learncomplicated tasks, such as making and using tools)

y The capacity to flourish (the ability to satisfy species-specific needs)y Sociability (being a member of a community)

y Possession of a life (attributing value to life itself)

Ethical decision making

What weight should be given to each of the morally relevant features in considering whether or

not research is acceptable? Are they factors to be weighed against human benefit? Should theybe understood as absolute constraints? For example, should any use of animals that are capable

of suffering be prohibited, or only the use of those that have higher cognitive capacities? Manypeople seem to support a hybrid approach. This involves a combination of laying down definite

limits for what should and should not happen (for example: animals with higher cognitive


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capacities, such as chimpanzees, should never be used in research) and weighing up the costsand benefits of a particular action (for example: research that causes minimum pain to a mouse

is acceptable if it helps to ascertain the safety of an important and frequently usedchemical).This approach can also be found in the Animals (Scientific Procedures) Act 1986: the

costs and benefits have to be weighed for each project and there are specific policies that prevent

the use of the Great Apes and the use of animals in the testing of new cosmetics.

The ethical debate comes down to disagreement on 2 questions:

1. What are the definite limits?

2. How do we weigh the different morally relevantfactors within the permitted limits?

To provide answers, we need to consider at least five further related questions:

1. What are the goals of research?

2. What is the probability of success?

3.

Which animals are to be used?4. What effect will there be on the animals used in the experiment?5. Are there any alternatives?

Ethical positions

After considering these questions, there was no single view to which they could all subscribe,thus reflecting the range of views that exists in society. Instead, we describe four possible ethical

positions, which represent points on a continuum. The report does not advocate any oneviewpoint as right. Rather, the reader is invited to decide which they find to be the most

acceptable.

The anything goes view: If humans see value in research involving animals, then it requires nofurther ethical justification.

The on balance justification view: Research involving animals is morally acceptable if the

costs are outweighed by the benefits, but every reasonable step must be taken to reduce the harmto animals.

The moral dilemma view: Most forms of research involving animals pose moral dilemmas.Animal research is morally unacceptable, but so is avoiding research that could be beneficial to

humans or animals.

The abolitionist view: There is no moral justification for any harmful research on animals thatis not to the benefit of the individual animal. Humans experiment on animals not because it is

right but because they can.


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ANIMALS USED

Many different animals are used for animal testing namely:

Mice (66%)

Mice (Mus musculus) have been domesticated for thousands of years and are easily the most

widely used vertebrate. They are mammals and are small, prolific, and easy to maintain.There are also a wide range of mutants such as obese,SCID, nude, hairless and dwarf mice.It is relatively easy to produce transgenic mice by injecting DNA into fertilised eggs, and there is

an extensive technology to inactivate specific genes either altogether or in specific tissues or atspecific times.There is strong genetic linkage homology between mice and humans so that once a

gene location is known in mice it's position can be predicted in humans. Mice are widely used infundamental research, but are also used in applied research such as in toxicity testing and drug

development, although the rat is generally preferred by toxicologists. The use of mice isincreasing at the expense of other species. The more that is known about the mouse, the more

valuable it becomes as a research animal.

Rats (18%)

Rats (Rattus norvegicus) have only been domesticated since the mid-eighteenth century and wereunknown in Europe until that time, having migrated from the far East. Rats are also small and

prolific, although they are substantially larger than mice. Although about 3.7 as many mice thanrats are used in research, this is not a direct reflection of their importance. Until recently more

papers were published using the rat than the mouse.

Strains of mice and rats:

Isogenic strainsIsogenic strains include inbred strains produced by many generations of brother x sister mating,

and the F1 (first generation) offspring of a cross between two inbred strains. They are likeimmortal clones of genetically identical individuals. The same genotype can be reproduced

indefinitely, though over a period of time there may be some genetic drift due to theaccumulation of new mutations.

Mutant strains

Many mutations have arisen in mouse and, to a lesser extent, in rat colonies either spontaneouslyor as a result of irradiation or chemical treatment. These have been preserved for research. Forexample mutant athymic "nude" mice and rats have a defective cell mediated immune system

and will grow human tumour xenografts. Various obese mutations have made a fundamentalcontribution to our understanding of obesity and metabolism.

Transgenic strains

These strains are made by the incorporation of DNA from another source into the genome. This


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is usually done by microinjection into an early embryo. The aim is usually to get an animal toexpress a foreign gene, over-express a gene, or express a gene in an abnormal tissue or at an

abnormal time. It is beyond the scope of this web to discuss the many uses for such strains.

"Knockout" strainsThese are strains in which one or more host genes have been inactivated either altogether or in aparticular tissue or at a particular time. They are widely used in fundamental research. A full

discussion of their properties is beyond the scope of this web.

Fish (6%)

Fish (several species) are overwhelmingly used in fundamental studies. Although there is nobreakdown by species in UK statistics, a large proportion are probably Zebra fish which are

widely used in fundamental studies of development. The species is oviparous and the egg anddeveloping embryo is transparent so that development can be observed directly in the live

animal. Trout are also used in toxicity testing.

Birds (4%)Chickens (Gallus domesticus) account for the vast majority of birds used in research, and most of

these are used in applied studies in veterinary medicine. A large proportion of these are probablyembryonated eggs which are counted in the UK once they reach half the incubation period.

These are widely used to culture viruses.

Guinea-pigs (1%)

Most of these are used in applied studies in human medicine.

Other rodents (1%)These include hamsters, gerbils and others. These seem to be used when no other species is

found to be suitable.

Dogs (0.2%)

Most of these are beagles which are used in applied studies in human medicine, probably largelyin toxicity testing and drug development.

Non-human primates (0.1%)

About three quarters are Old-World monkeys such as Macaques, with about a quarter of them areNew-World monkeys such as marmosets. A large majority of them are used in applied studies in

human medicine, and in particular in toxicity testing. Much smaller numbers are used inacademic research, some of which involves neuroscience and the study of diseases such as

Parkinson's disease which can not always be adequately modelled in rodents.


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All other speciesThese include sheep (0.7%), pigs (0.4%), rabbits (0.6%), amphibians (0.3%) and others (0.2%).

It is difficult to generalise about why they are used. However, sheep and pigs are often used in

surgical studies because of their large size and ready availability, rabbits are probably largelyused for the production of polyclonal antibodies, and amphibians are mostly used in fundamentaldevelopmental research.

The feed for the mice and rats included:

Crude protein 22.1%

Crude oil 4.31%

Crude fibre 3.15%

Ash 5.11%

Sand silica 1.2%

Aflatoxin 0%

Energy 3620 kcal/kg.

Rabbits used- Oryctolagus cuniculus (European Rabbit).

The feed used for rabbit:

Crude protein 22.15%

Crude oil 4.12%

Crude fibre 12.1%

Ash 5.11%

Sand silica 1.15%

Aflatoxin 0%

Energy 3626 kcal/kg.


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RESTRAINT AND HANDLING OF ANIMALS

GENERAL PRINCIPLES

The use of proper restraint and handling techniques reduces stress to animals and also to the

researcher. Handling stress represents an experimental variable and should be minimizedwhenever possible. Animals can inflict serious injuries to humans and to themselves as a result

of improper handling.

y Animals experience stress as a result of shipping. All large animals must be allowed toacclimate to the facility for three days. During this time they may not be experimentally

manipulated. Acclimation periods of up to one week are recommended for all animals.y If a study will involve significant handling of animals it is recommended that the animals

be acclimated to the handling. Prior to experimental manipulation, handle the animal on a

regular basis in a non-threatening situation, e.g. weighing, petting, giving food treats.Most animals, even rodents will respond positively to handling and will learn torecognize individuals.

y Handle animals gently. Do not make loud noises or sudden movements that may startlethem.

y Handle animals firmly. The animal will struggle more if it sees a chance to escape.y Use an assistant whenever possible.

y Use restraint devices to assist when appropriate.y Chemical restraint should be considered for any prolonged or potentially painful

procedure.

HANDLING METHODS

The methods described below will assist with performing basic manipulations. Alternate

techniques may be needed for special procedures. Most of these methods are also demonstratedin video tapes available to investigator. An excellent website containing laboratory

biomethodology for rodents and rabbits is also available with descriptions and pictures of drugadministration, blood collection and sex determination.

Raising Antisera in rabbits:

1. Female rabbits,( 2-2.5kg) are used.2. Priming immunization: Injection mixture of homogenized 500l of the specific antigen

with 500l complete Freund's adjuvant (CFA) solution using repeated aspiration with 18gasyringe is prepared and Subcutaneous/Intramuscular injection using 1ml syringe and 23

1/2ga needle is given.


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3. Pre-immune blood sampling: Anaesthesia (ketamine [17.5mg/kg] + xylazine [2.5mg/kg]mixture) given. Swab the site of injection with alcohol prior to injection. Rabbit bled via

central auricular artery (5ml) using 25 3/4ga wing infusion set.4. First booster immunization: After 3 weeks, the rabbit is bled to check its antibody response

to the priming immunization and then given its first booster immunization dose (same

dosage as the priming immunization but with incomplete Freund's adjuvant(IFA) instead).5. All bleeding is done with prior anaesthesia given to the rabbit.6. Further booster immunizations can be given at 3-week intervals till the final harvest. The

optimal number of booster immunizations to be given before final harvest is obtained byplotting an antibody titer curve.

7. Final harvest is done by bleeding a whole blood volume of 80ml (or maximum volume thatcan be obtained) and the rabbit is then culled with injection of euthanasia into the marginal

ear vein. At National toxicology centre we injected rabbits with commercially availableantigen O and H ofSalmonella typhi.

Marginal vein of the rabbit used at NTC for bleeding purposes.

Common routes of drug administration to laboratory animals include:

1. Gastrointestinal1. Oral (per os) - through the mouth2. Gavage - into the stomach via a tube or gavage needle

3. Rectal (per rectum) - into the rectum via the anus

4.

NPO (nil per os) - nothing by mouth. Usually prescribed prior to generalanesthesia.

2. Parenteral

1. Intravenous (IV) - directly into the venous bloodstream2. Intraperitoneal (IP) - into the abdominal cavity

3. Subcutaneous (SC) - under the skin4. Intramuscular (IM) - into a muscle

5. Intradermal (ID) - into or between layers of skin


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6. Intrathecal (IT) - into the subarachnoid space of the spinal cord7. Intracranial (IC) - into the substance of the brain

The route selected for drug administration is governed by the nature of the agent beingadministered, the animal, the purpose of administration, and other factors. The techniques for

each route vary from species to species, but all require a general understanding of local anatomyat the injection site

In the NTC, we got to handle the mice. The mice were injected with water via four differentroutes namely intramuscular, intraperetonial, intravenous and oral routes. They alsodemonstrated us with the technique of collection of blood through renal route, cervical

dislocation and dissection.

We also handled rabbits for raising antibodies against Salmonella O and H antigen. The rabbitwas injected with the commercially available antigen. The blood was collected from the marginal

ear lobe. This blood was further used for antibody preparation.


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In all it was an excellent experience and I look forward for more experience in handling theanimals as I have learnt that from this that practice is needed for proper accurate handling of

animals without hurting them.

nikhil joshi ntc

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