ANIMAL MODELS IN TOXICOLOGICAL STUDIES

PRESENTED BY

Dr. SINDHU. K

MVSc SCHOLAR,

DEPT OF VPT,

POOKODE, WAYANAD.

ANIMAL

(According to ANIMAL WELFARE ACT, USA )

• Animal is defined as any live or dead dog , cat , non human primate , guinea pig , hamsters , rabbit or any other warm blooded animal , which is being used or is intended for use for research , teaching , experimentation , exhibition or as a pet.

ANIMAL MODEL

• An animal model is defined as a specific combination of an animal species, challenge agent and route of exposure that produces a disease process or pathological condition that in multiple important aspects corresponds to the human disease or condition of interest.  

• In the context of animal model qualification, the model-defining natural history studies are the animal studies that establish the ranges of values of key parameters of the disease or condition that will be specified in the context of use for the qualified model and that will be used as measures of quality control and quality assurance when the model is replicated.

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CLASSIFICATION OF ANIMAL MODELS

1. EXPERIMENTAL : A model in which an experimenting induced

conditions mimic a human disease

2. NEGATIVE : A model in which particular condition cant be

produced , & is therefore studied to better understand the reason for the protective or resistant affects

3. SPONTANEOUS : A model in which the animal naturally develops

a disease or some other conditions of interest

4. ALTERNATIVE MODEL is defined as technique that reduces or

eliminates the need for live animals & there by prevents potential pain & distress in animals

‘• Computer models and cell

cultures are good for screening and are used frequently.• Such models cannot replicate

complicated interactions in the whole system.• Final testing depends on studies

in animals; sometimes it is required by law.• Animal and non-animal models

used in conjunction achieve the best answer.

WHAT IS ANIMAL TESTING?

• The term "animal testing" refers to procedures performed on living animals for purposes of research into basic biology and diseases, assessing the effectiveness of new medicinal products, and testing the human health and/or environmental safety of consumer and industry products such as cosmetics, household cleaners, food additives, pharmaceuticals and industrial/agro-chemicals.

• All procedures, even those classified as “mild,” have the potential to cause the animals physical as well as psychological distress and suffering.

• Often the procedures can cause a great deal of suffering. Most animals are killed at the end of an experiment, but some may be re-used in subsequent experiments.

CAN ANIMAL MODELS OF DISEASE RELIABLY INFORM HUMAN STUDIES?

 

"The value of animal experiments for predicting the effectiveness of treatment strategies in clinical trials has remained controversial, mainly because of a recurrent failure of interventions apparently promising in animal models to translate to the clinic”

EVIDENCES

Polio

• Landsteiner and Popper proved it infectious; able to transmit disease to monkeys.

• Salk and Sabin developed their vaccine through work with chickens and monkeys.

Diabetes

• Banting and Best showed importance of insulin in dogs.

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Infant Mortality• Studies in sheep and lambs led to use of steroids in treatment of

respiratory distress syndrome (formerly hyaline membrane disease), a major cause of death in premature infants.• Advances in understanding and treatment of sudden infant death

syndrome (SIDS) came from studies in rats, mice, dogs, and sheep

Cystic Fibrosis• A major killer of young adults.

• Mouse models led to understanding role of chloride channels.

• Genetic therapies on the horizon are an outgrowth of work in mice.

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High Blood Pressure (HBP)• Goldblatt linked HBP to kidney in rats, cats, and dogs; led to diuretics

and angiotensin converting enzyme inhibitors to treat high blood pressure.

• Cushing linked HBP to brain in dogs; led to understanding sympathetic nervous system influence on blood pressure and drugs to treat it.

Obesity• Major risk factor for diabetes mellitus, high blood pressure, heart

attack, stroke and certain cancers. • Epidemic in the United States: 64% of adults are overweight and 25%

are obese.• Mouse models and Zucker obese rats shedding new light on causes of

overeating, importance of leptin receptors, and ways that obesity leads to disease.

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Bioterrorism• Smallpox vaccine from calves

• “Two animal rule” – FADA mandates that all vaccines must be tested for efficacy and safety in two animals (typically rodent and non-human primate) before introduction in humans

• Botulinum antitoxin tested in mice and non-human primates

AIDS• Numerous animal models in studies to understand the disease and how

it attacks the immune system.• Current anti-AIDS treatment developed in animal models have greatly

extended life expectancy and quality of life for AIDS victims.• AIDS vaccines being developed in monkeys.

HISTORY

• The earliest references to animal testing are found in the writings of the Greeks in the 2nd and 4th centuries BCE.

•  Aristotle and Erasistratus were among the first to perform experiments on living animals.

•  Galen, a physician in 2nd-century Rome, dissected pigs and goats, and is known as the "father of vivisection".

• An Experiment on a Bird in an Air Pump,

from 1768, by Joseph Wright

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• In the 1880s, Louis Pasteur convincingly demonstrated the germ theory of medicine by inducing anthrax in sheep.

• In the 1890s, Ivan Pavlov famously used dogs to describe classical conditioning.

• Insulin was first isolated from dogs in 1922, and revolutionized the treatment of diabetes.

• On November 3, 1957, a Soviet dog, Laika, became the first of many animals to orbit the earth.

• In the 1970s, antibiotic treatments and vaccines for leprosy were developed using armadillos, then given to humans.

•  The ability of humans to change the genetics of animals took a large step forwards in 1974 when Rudolf Jaenisch was able to produce the first transgenic mammal, by integrating DNA from the SV40 virus into the genome of mice.

•  This genetic research progressed rapidly and, in 1996, Dolly the sheep was born, the first mammal to be cloned from an adult cell.

CLAUDE BERNARD

REGARDED AS THE "PRINCE OF VIVISECTORS",

ARGUED THAT EXPERIMENTS ON ANIMALS ARE "ENTIRELY

CONCLUSIVE FOR THE TOXICOLOGY AND HYGIENE OF

MAN"

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•  Claude Bernard—who is sometimes known as the "prince of vivisectors" and the father of physiology, and whose wife, Marie Françoise Martin, founded the first anti-vivisection society in France in 1883

• In 1822, the first animal protection law was enacted in the British parliament, followed by the Cruelty to Animals Act (1876), the first law specifically aimed at regulating animal testing. The legislation was promoted by Charles Darwin

• Opposition to the use of animals in medical research first arose in the United States during the 1860s, when Henry Bergh founded theAmerican Society for the Prevention of Cruelty to Animals (ASPCA), with America's first specifically anti-vivisection organization being the American AntiVivisection Society (AAVS), founded in 1883

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• Toxicology testing became important in the 20th century.

• In the 19th century, laws regulating drugs were more relaxed. For example, in the U.S., the government could only ban a drug after a company had been prosecuted for selling products that harmed customers.

• However, in response to the Elixir Sulfanilamide disaster of 1937 in which the eponymous drug killed more than 100 users, the U.S. congress passed laws that required safety testing of drugs on animals before they could be marketed. Other countries enacted similar legislation.

•  In the 1960s, in reaction to the Thalidomide tragedy, further laws were passed requiring safety testing on pregnant animals before a drug can be sold

REGULATIONS

• Licensing

• Registration

• Research facilities

• Attending veterinarian & adequate veterinary care

• Identification of animals

• Records

• Compliance with standards & holding period

STANDARDS

• Specification for human handling care

• Treatment & transportation of experimental animals

INDIAN MEDICAL ORGANIZATIONS

To ensure uniform quality of clinical research by Good Clinical Practices throughout the country and to generate data for registration of new drugs before use in the Indian population.

CSIR

• Council of Scientific and Industrial Research• is an autonomous body and India's largest Research and

Development (R&D) organization.

CDRI

• Central Drug Research Institute• CDRI is the laboratory functioning under the aegis of the

council of scientific and Industrial Research of India.

SAFETY & CLINICAL

DEVELOP

MENT--

TOXICOLO

GY

• Toxicology group is involved in profiling of candidate drugs according to schedule Y guidelines. Systemic toxicology, reproductive toxicology, genetic toxicity, immunotoxicity, local toxicity and carcinogenicity are being done for NCEs.

INDIAN REGULATIONS

• The Government of India has authorized the National Accreditation Board of Testing and Calibration Laboratories (NABL), promoted by the Department of Science andTechnology, to provide accredition services to laboratories covering a wide range of subjects including biological and clinical laboratories.

• The NABL is a full member of the International Laboratory Accreditation Cooperation and the Asia Pacific Laboratory Cooperation.

• Such accreditation of animal facilities would demonstrate their commitment to responsible animal care and use and good science since such an accreditation is an indicator of an institution’s ability to comply with its assurances.

INDIAN NATIONAL SCIENCE ACADEMY• This report was submitted by the Expert Committee Constituted by the

Indian National Science Academy, New Delhi

• The need to maintain the animals under standard animal husbandry conditions, to handle them gently and humanely, and limit unnecessary usage by strictly enforcing ethical considerations demands utmost attention.

• A Committee was constituted to consider the various issues involved in the ethical use of Animal Experimentation, and the Committee besides other recommendations updated “Guidelines”.

• Published by S. K. Sahni, Executive Secretary, Indian National Science Academy, Bahadur Shah Zafar Marg, New Delhi 110 002 and printed at Bengal Offset Works, 335, Khajoor Road,Karol Bagh, New Delhi 110005

Tel : 3524200, 3610455, 7774614

INSA GUIDELINES

• In India, the need to develop guidelines for the use of animals in research has been discussed at various forums. Unfortunately no standard document was available for reference till 1992 when the Indian National Science Academy developed the guidelines for use of animals in scientific research. Considering the knowledge generated internationally over the years and the guidelines of WHO,NIH associated NRC, USA and European Union, the INSA guidelines have been updated.

• The INSA guidelines are the only ones available at the National level and adopted by well established institutions in India for care and use of their laboratory animals

LEGAL PROVISION

• The Prevention of Cruelty to Animal Act of 1960 has provided the constitution of a committee under the Animal Welfare Board to control and supervise experiments on animals.

• It has also provided inspection of all animal holdings through designated inspectors and the institutions not abiding by the standard requirements can be prosecuted. The relevant sections of the act can be obtained in Chapter IV

• THE PREVENTION OF CRUELTY TO ANIMALS ACT, 1960 (59 OF 1960)

As amended upto 30th July, 1982

CHAPTER IV

EXPERIMENTATION OF ANIMALS

EUROPE

• Experiments on vertebrate animals in the European Union are subject to Directive 86/609/EEC on the protection of Animals used for Experimental and other scientific purposes, adopted in 1986

• In November 2010, "Directive 2010/63/EU on the protection of animals used for scientific purposes", which updates and replaces the 1986 Directive 86/609/EEC, was finalized and came into force. Full implementation of the new EU directive starts January 1, 2013

FRANCE

• In France, legislation (principally the decree of October 19, 1980) requires an institutional and project license before testing on vertebrates is carried out. An institution must submit details of their facilities and the reason for the experiments, after which a five-year license may be granted following an inspection of the premises. The project licensee must be trained and educated to an appropriate level. Personal licenses are not required for individuals working under the supervision of a project license holder. These regulations do not apply to research using invertebrates

UNITED KINGDOM

• The types of institutions conducting animal research in the UK in 2004 were:

• universities (42.1%);

• commercial organizations (33.3%);

• non-profit organizations (4.9%);

• government departments (2.4%);

•  National Health Service hospitals (0.9%);

• public health laboratories (0.6%);

• other public bodies (15.8%).

CONTD…,

• During 2002 House of Lords select committee inquiry into animal testing in the UK, witnesses stated that the UK has the tightest regulatory system in the world, and is the only country to require a cost-benefit assessment of every licence application.There are 29 qualified inspectors covering 230 establishments, which are visited on average 11–12 times a year.

• A campaign document by Animal Aid alleges that the Animals (Scientific Procedures) Act 1986 is a "vivisectors' charter," allowing researchers to do as they please and making them practically immune from prosecution. The document claims that licences to perform experiments are obtained on the basis of a "nod of approval" from the Home Office Inspectorate, and that the Home Office relies on the researchers' own opinions of the cost-benefit assessment regarding the value of the experiment versus the amount of suffering it will cause

GERMANY

• The German Animal Welfare Act is designed to enforce the utilitarian principle that there must be good reason for one to cause an animal harm and identifies that it is the responsibility of human beings to protect the lives and well-being of their fellow creatures. There are thirteen sections in the Animal Welfare Act, each containing Articles that go into detail of the specific sections.

JAPAN• The system in Japan is one of self-regulation; there are no

regulations like Western countries, only the 3Rs principle are written into the Law for Humane Treatment and Management of Animals.

• This law was amended in June 2005 and enforced in June 2006. The Management of Animals is responsible for administering the newly amended law.The amendment of this law was the conceptual idea of self-regulation and not being restricted by legislative constraints, it was approved by the members of the Japanese Diet who saw that care for laboratory animals and the use of laboratory animals are two different concepts that were concerned with science and animal welfare, respectively.

• This law requires those using animals to follow the principles outlined in the 3Rs

3 R ` S

3 R`S

• The 3Rs principle of animal experimentations was introduced to the world by Russell and Burch in 1959.

•  In terms of ethics in animal experimentation, it gave detailed information on the guidelines that were formulated through the concerns of the ministries and the Science Council of Japan.

• The 3Rs: Replacement, Reduction, and Refinement, which are also known as "the standards relating to the care and management of laboratory animals and relief of pain", are covered in a detailed guideline based on the current law.

CHARACTERISTICS OF REGULATIONS ON ANIMAL EXPERIMENTS IN JAPAN

• Laws specify the responsibility of the owner of the animal.

• Laws call for 3Rs emphasizing the alleviation of pain and distress as well as humane death of animals used for scientific purposes.

• Administrative guidance encourages the ethical use of animals.

• Self-regulation system similar to the US and Canada.

• Recommendation for designating Institutional Animal Care and Use Committee (IACUC).

• Exemption from legal registration/inspection.

IACUC

• In 1985 the 3Rs were outlined into 11 principles by the Council of International Organizations for Medical Sciences (CIOMS) which have become the international standard to govern animal experimentation.

• Japanese law is based on the idea of a self-regulation system for the use of animals in laboratory experimentation.

• Due to this, notification only recommends designation of an Institutional Animal Care and Use Committee (IACUC). Although not required by law almost all pharmaceutical companies as well as medical schools have established IACUCs; livestock and laboratory animal facilities are exempt from registration with the IACUC and legal inspection

UNITED STATES

• In the United States, animal testing on vertebrates is primarily regulated by the Animal Welfare Act of 1966 (AWA), and the Animal Welfare Regulations which is enforced by the Animal Care division of the Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA).

• The AWA contains provisions to ensure that individuals of covered species used in research receive a certain standard of care and treatment, provided that the standard of care and treatment does not interfere with "the design, outlines, or guidelines of actual research or experimentation

CANADA

The federal government does not have jurisdiction to pass laws that involve experiments on animals. The provinces have jurisdiction concerning that area. The federal government, however, is involved in three areas: the criminal law power, the health power, and the spending power.

• The Criminal Code of Canada Section 446 and 447 of the Criminal Code protect animals from cruelty, abuse and neglect. This section of the Criminal Code has been under review for several years.

• The Health of Animals Act The Health of Animals Act (1990) and its regulations are aimed primarily at protecting Canadian livestock from a variety of infectious diseases that would threaten both the health of the animals and people, and Canadian trade in livestock with other countries. This act is used both to deal with named disease outbreaks in Canada, and to prevent the entry of unacceptable diseases that do not exist in Canada.

CONTD

• The Spending Power : the imposition of CCAC standards on facilities receiving funding from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council. Where the government itself awards a contract on an academic or non-academic institution, clause A9015C of Public Works Standard Acquisition Clauses and Conditions Manual imposes conditions related to the care and use of experimental animals in public works and government services  

AUSTRALIA

• In Australia, Animal Ethics Committees (AECs) determine whether the use of an animal is valid or not. AECs must follow the Code in order to ensure the wellbeing of the animals used for research. The Code emphasizes the responsibilities of investigators, teachers and institutions using animals to:

• ensure that the use of animals is justified, taking into consideration the scientific or educational benefits and the potential effects on the welfare of the animals

• The researchers must submit a written proposal to an AEC stating what is to be accomplished, a defense for the study, and the ethical and wellbeing of the animals used reflecting the 3Rs.

NEW ZEALAND

• New Zealand’s Animal Welfare Act 1999 requires owners and people in charge of animals to ensure the physical, health and behavioural needs of animals are met, and that pain and distress are alleviated.

• In New Zealand, as in many countries, laboratory animals (mainly rodents) and farm animals (mainly cattle and sheep) are used in research, testing and teaching – commonly referred to as RTT. Animal use in RTT is strictly controlled under the Animal Welfare Act 1999 and organisations using animals must follow an approved code of ethical conduct.

• The Ministry for Primary Industries (MPI) administers the Act and leads animal welfare policy and practice in New Zealand. The National Animal Ethics Advisory Committee (NAEAC) was established under the Animal Welfare Act to provide independent advice to the Minister for Primary Industries

BRAZIL

• The federal law for the scientific use of animals was passed in 2008. The law established the National Council for the Control of Animal Experimentation (CONCEA) and demanded that institutions create an ethics committee on the use of animals.

• In 2009, Decree 6899/2009 defined CONCEA as the governing and advisory body, under the Ministry of Science and Technology, to authorize accreditation to registered institutions and to license those institutions to use animals in research. The same decree also states that an electronic database be developed to allow breeding and research facilities to register in order to apply for CONCEA accreditation.

• Brazil also reinforces the 3Rs.

GUIDE FOR THE CARE & USE OF LABORATORY ANIMALS

1) INSTITUTIONAL POLICIES

•Monitoring the care & use of animals

• Personal qualifications

• Personal hygiene

•Occupational health

• Experiments involving hazardous agents

• Special consideration

2) LABORATORY ANIMAL HUSBANDRY

•Housing

• Animal environment

• Food , water & bedding

• Sanitation

• Identification & record keeping

• Emergency , weekend & holiday care

3) VETERINARY CARE

• Preventative medicine

• Surveillance , diagnosis , treatment & control of diseases

• Anesthesia & analgesia

• Surgery & post surgical care

• Euthanasia

4) PHYSICAL PLANTS

• Physical relationship of animal facilities to laboratories

• Functional areas

• Construction guidelines

• Aseptic surgery

ANIMAL PAIN

• In accordance with the AWA

• guide for the care & use of laboratory animals

• public health service policy for the human care

•Use of laboratory animals

Veterinarians & investigators must identify , eliminate sources of pain & distress , with the exception of those procedures that are essential to research in question & approved by the IACUC

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TERMINOLOGIES

COMFORT

A state of physiological , psychological & behavioural equilibrium in which an animal is accoustomed to its environment & engages in normal activities – feeding , drinking , grooming , social interaction , sleeping waking cycles & reproduction

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WELL BEING

A positive mental state that reflects the level of welfare & comfort of an animal

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DISCOMFORT

A minimal change in an animal`s adaptive level or baseline state as a result of changes in its environmental or biological , physical , social or psychotic alterations

Physiological or behavioural changes that indicates a state of stress might be observed , but are not marked enough to indicate distress

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STRESS

The effect produced by external events or internal factors referred to as STRESSORS , which include an alteration in an animal`s biological equilibrium

DISTRESS

An adverse state in which an animal is unable to adapt completely to stressors & the resulting stress & shows maladaptive behaviours

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ANXIETY & FEAR

Emotional states that are traditionally associated with stress. They can be adaptive in that they inhibit an organisms actions that could lead to harm or cause it to act in ways allowing it to escape from potentially harmful situations

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PAIN

Results from potential or acute tissue damage

Pain can be considered a potent source of stress , that is , A STRESSOR.it can also be considered a state of stress itself & can lead to distress & malabsorptive behaviours

CRITERIA`S TO BE CONSIDERED FOR SELECTION OF LIVE ANIMAL EXPERIMENT

• Procedure should yield results beneficial to well being

• The species & no of animals should be appropriate for experimental purpose

• The proposed procedures shouldn’t unnecessarily duplicate previous experiment

• Appropriate analgesics , anesthetics & tranquilizing drugs to be used to minimize pain & discomfort

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•Methods of euthanasia

• The individuals performing the experimental procedure & caring for the animals need to be properly trained

In general procedures that causes minimal pain or discomfort to humans & place the animals in minimum distress are considered ACCEPTABLE

ANIMAL NUTRITION

• All animals require regular amount of clean pure water & food

• Complete balanced diet or certified diets are preferred for research purposes

• Most toxicological studies employ ad libitum feeding conditions

In which animals are allowed to regulate their own dietary intake to meet energy requirements

• Ad libitum feeding for long term rodent bio-assay impacts longevity , carcinogenesis & over all animal health

FOOD & WATER REQUIREMENTS

SPECIES DAILY FOOD REQUIREMENT DAILY WATER REQUIREMENT

Mouse 3 – 6 g 3 – 7 ml

Rat 10 – 20 g 20 – 30 ml

Hamster 7 – 15 g 7 – 15 ml

Guinea pig 20 – 30 g 12 15 ml / 100 g

Rabbits 75 – 100 g 80 – 100 ml / kg

Cat 100 – 225 g 100 – 200 ml

Dog 250 – 1200 g 100 – 400 ml / day

Primate 40 g / kg 350 – 1000 ml

FASTING

• Like humans , animals are often fasted in preparation for blood collection

Non human primates , G pig : 18 – 24 hrs

Mice : 4 – 6 hrs

ANESTHESIA & ANALGESIA

• Investigators using live animals must employ appropriate anesthetic , analgesic , sedative agents necessary to control pain & distress , unless use of such agents would interfere with specific objectives of the research

• Most of the PAIN RELIEVING AGENTS are controlled substances & must be ensured that drug supplies are adequately protected & inventoried in accordance with the requirement of the statute

GENERAL PRINCIPLES REGARDING ANESTHESIA , ANALGESIA & TRANQUILIZATION

• The effect of technique on experimental objectives , including drug interactions & interferences with test substances say competing metabolic pathways

• The health of animal should be carefully evaluated before instituting any tranquilizing procedure

• The specific drug selected selected should provide minimal level of CNS depression necessary

STAGES OF ANESTHESIA

STAGE 1 ANESTHESIA

• Involuntary excitation

•Dilation of pupils

• Possible increased salivation

• Loss of consciousness

• Spontaneous voiding of urine & feces

2) STAGE 2 ANESTHESIA

• BREATHING pattern becomes more rapid & shallow

• Orbital movement of eyes becomes apparent ( NYSTAGMUS )

3) STAGE 3 ANESTHESIA

• Referred to as surgical stage anesthesia & is sub divided into 4 different PLANES of progressively deeper unconsciousness

• Plane 2 is routinely used for minor surgery & for some major surgical procedures

PLANE 1

• Muscle tone is decreased

• Pedal reflex disappears but Palpebral & corneal reflexes remains unchanged

• Pulse rate & blood pressure are usually normal

PLANE 2

• Pupil constricts & palpebral reflex absent

• Laryngeal reflex diminishes

• Salivation stops

• Corneal reflex remains intact

PLANE 3

• Corneal reflex disappears completely

• Breathing becomes abdominal & slow

• Heart rate may decrease

PLANE 4

• Pupils dilate

• Thoracic breathing stops

• Death follows

COMMONLY USED ANESTHETIC & TRANQUILIZING AGENTS IN ANIMAL MODEL STUDIES

• Atropine as pre anesthetic

• Acepromazine + ketamine

• Diazepam – schedule IV drug { Anticonvulsant properties }

• Narcotic agents : hypnotic & analgesic effects with resulting depression of cardiovascular & thermoregulatory systems

Morphine , meperidine , etorphine , fentanyl

• Innovar – vet : narcotic analgesicfentanyl @ 0.4 mg / ml + tranquilizer droperidol @ 20 mg / ml

• Ketamine : state of chemical restraint & anesthesia but reflex remains constant. Wide margin of safety, short duration with recovery, minimal adverse effects

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• Pentobarbital , thiamylal & cholarlhudrate others with small margin of safety

INHALANT ANESTHETICS

• DIETHYL ETHER : Although it provides good analgesia & muscle relaxation , vapors irritate the respiratory mucosa

extremely flammable & explosive

• HALOTHANE : highly volatile requires a vapourizer to produce precise concentrations

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• METHOXY FLURANE : non explosive inhalant , low volatility produces good analgesia & muscle relaxation

• ISOFLURANE : non flammable , non explosive general inhalant anesthetic agent

• NITROUS OXIDE : potent inhalant anesthetic , nonirritating , non explosive , often used in conjunction with other agents

CONTRAINDICATIONS

Mouse : chloroform

Cats : morphine

G pigs : innovar – vet i/m inj

Rats : methoxyflurane

EUTHANASIA

• Methods of euthanizing laboratory animals are chosen to induce rapid unconsciousness and humane death without pain or distress.

•  The methods that are preferred are those published by councils of veterinarians.

• The animal can be made to inhale a gas, such as carbon monoxide and carbon dioxide, by being placed in a chamber, or by use of a face mask, with or without prior sedation or anesthesia

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• PHYSICAL EUTHANASIA : cervical dislocation & decapitation recommended only when scientifically justified

Cervical dislocation is considered humane for poultry , mice , rats weighing less than 200 g & rabbit weighing less than 1 kg

• VERIFICATION OF DEATH : irregardless of specific euthanasia methods used , it is imperative that death be verified by examining the animal for cessation of vital signs

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REFERENCES

• CRC handbook on toxicology

• Wikipedia : animal models

• Google images

THANK YOU