Dr. Pravin R.

Tirgar CPCSEA Guideline

Research Methodology

Dr. PRAVIN TIRGAR

PROFESSOR, SCHOOL OF PHARMACY, RK
UNIVERSITY
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CPCSEA
Committee for the Purpose of Control and Supervision of Experiments on
Animals
The motto of Prevention of Cruelty to Animals (PCA) Act 1960 as amended in 1982, is to
prevent infliction of unnecessary pain or suffering on animals. The Central Government has
constituted a Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) which is duty bound to take all such measures as may be necessary to ensure that
animals are not subjected to unnecessary pain or suffering before, during or after the
performance of experiments on them. For this purpose, the Government has made "Breeding
of and Experiments on Animals (Control and Supervision) Rules, 1998" as amended during 2001
and 2006, to regulate the experimentation on animals.

Goals of CPCSEA:
1. Assure Good Laboratory Practice (GLP) for animal facilities

2. Registration of establishments conducting animal experimentation or breeding of animals for

this purpose.

3. Selection and appointment of nominees in the Institutional Animal Ethics Committees of

registered establishments.

4. Approval of Animal House Facilities on the basis of reports of inspections conducted by CPCSEA.

5. Permission for conducting experiments involving use of animals.

6. Recommendation for import of animals for use in experiments.

7. Action against establishments in case of violation of any legal norm/stipulation.

8. Standard Operating Procedures (SOP) for Institutional Animal Ethics Committee (IAEC) of the
CPCSEA.

9. Mandatory Actions for All Establishments Registered with CPCSEA.

Most IPM

3-R Concept - 3Rs – THE ETHICAL USE OF ANIMALS

1. Replacement: methods which avoid or replace the use of animals in research

2. Reduction: use of methods that enable researchers to obtain comparable levels of information
from fewer animals, or to obtain more information from the same number of animals.

3. Refinement: use of methods that alleviate or minimize potential pain, suffering or distress,
and enhance animal welfare for the animals used.

Replacement > Reduction > Refinement

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Veterinary care
Adequate veterinary care must be provided and is the responsibility of a veterinarian
or a person who has training or experience in laboratory animal sciences and
medicine.

Following veterinary care carried out on regular basis by in charge of animal house:

1. Daily observation of animals and animal house facilities

2. Note timely and accurate information on problems in animal behaviors and health
status.

3. Supervising animal nutrition and sanitation

4. Ensure air-conditioning, exhaust fan and cleanliness in animal house

5. Monitoring hazard conditions, chemicals and drug used on animals

6. Take care of usage on animal for research/academic protocol

7. Ensure quarantine when infection to animal or new animals in animal house

8. Make policy, procedure for veterinary care like reviewing protocol, proposals,
animal husbandry and welfare.

9. Give necessary treatment in case of illness to animals.

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Quarantine:
“Quarantine is the separation of newly received animals from those already in
the facility until the health and possibly the microbial status of the newly received
animals have been determined.”

An effective quarantine minimizes the chance for

introduction of pathogens into an established colony.

The duration at quarantine in.

 Small lab animals from one week to one month and large animals allowed up to 6
weeks (cat, dog, monkey, etc.)

 Effective quarantine procedures should be used for non-human primates to

help limit exposure of humans to zoonotic infections. The period varies from 2 to 3
months depending on the reaction of TB testing.

 Regardless of the duration of quarantine, newly received animals should be

given a period for physiologic, psychological and nutritional stabilization before their
use. The length of time stabilization will depend on the type and duration of animal
transportation, the species involved and the intended use of the animals.

 Physical separation of animals by species is recommended to prevent

interspecies disease transmission and to eliminate anxiety and possible physiological
and behavioral changes due to interspecies conflict.
Such separation is usually accomplished by housing different species in
separate rooms; however, cubicles, laminar-flow units, cages that have filtered air or
separate ventilation, and isolators shall be suitable alternatives.

 In some instances, it shall be acceptable to house different species in the same

room, for example, if two species have a similar pathogen status and are behaviorally
compatible.

 Separate set of personnel should be identified for taking care of these animals
and other people should be restricted from entering in to the facilities unless otherwise

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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required and after handling these animals they should not be handling any other
animals in the facilities.

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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SURVEILLANCE, DIAGNOSIS, TREATMENT AND CONTROL OF

DISEASE:

All animals should be observed for signs of illness, injury, or abnormal

behavior by animal house staff. As a rule, this should occur daily, but more-frequent
observations might be warranted, such as during postoperative recovery or when
animals are ill or have a physical deficit. It is imperative that appropriate methods be
in place for disease surveillance and diagnosis (Annexure 1 & 2).

Postmortem examination and signs of illness, distress, or other deviations from

normal health condition in animals should be reported promptly to ensure appropriate
and timely delivery of veterinary medical care. Animals that show signs of a
contagious disease should be isolated from healthy animals in the colony. If an entire
room of animals is known or believed to be exposed to an infectious agent (e.g.
Mycobacterium tuberculosis in non-human primates), the group should be kept intact
and isolated during the process of diagnosis, treatment, and control. Diagnostic
clinical laboratory may be made available.

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Personal Hygiene:
Animal care programs require technical and husbandry support. Institutions
should employ people trained in laboratory animal science or provide for both formal
and on-the-job training to ensure effective implementation of the animal care and
personal hygiene to prevent infection from animal to human and vice versa.

PERSONAL HYGIENE

 need to use apron, gloves, mask

 handling of detergents and other cleaning substances

 zoonoses (Different species) - need of safety handling

 antidotes for specific poisons if handling poisonous animals like venomous snakes
- first aid

It is essential that the animal care staff maintain a high standard of personal
cleanliness. Facilities and supplies for meeting this obligation should be provided
with appropriate Personnel Protective Equipment (PPE) e.g. showers, change of
uniforms, footwear etc.

Clothing suitable for use in the animal facility should be supplied and
laundered by the institution. A commercial laundering service is acceptable in many
situations; however, institutional facilities should be used to decontaminate clothing
exposed to potentially hazardous microbial agents or toxic substances.

It is acceptable to use disposable gloves, masks, head covers, coats, coveralls and shoe
covers. Personnel should change clothing as often as is necessary to maintain personal
hygiene. Outer garments worn in the animal rooms should not be worn outside the
animal facility.

Washing and showering facilities appropriate to the program should be

available. Personnel should not be permitted to eat, drink, smoke or apply cosmetics
and perfumes in animal rooms. They should finish the work with animals as early as

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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possible and sit somewhere else outside and not in the animal rooms / areas. A
separate area or room should be made available for these purposes.

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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LOCATION OF ANIMAL FACILITIES TO LABORATORIE

Good animal husbandry and human comfort and health protection require
physical separation of animal facilities from personnel areas such as offices, break
room, training and education room.

Laboratory animals are very sensitive to their living conditions. It is important

that they shall be housed in an isolated building located as far away from human
habitations as possible and not exposed to dust, smoke, noise, wild rodents, insects
and birds. The building, cages and environment of animal rooms are the major
factors, which affect the quality of animals.

This separation can be accomplished by having the animal quarters in a separate

building, wing, floor, or room. Careful planning should make it possible to place
animal housing areas adjacent to or near laboratories, but separated from them by
barriers such as entry locks, corridors, or floors.

While planning an animal facility the space should be well divided for various
activities. The animal rooms should occupy about 50-60% of the total constructed
area and the remaining area should be utilized for services such as stores,
washing, office and staff, machine rooms, quarantine and corridors. The
environment of animal room (Macro-Environment) and animal cage
(Microenvironment) are factors on which the production and experimental
efficiency of the animal depends. Since animals are very sensitive to
environmental changes, sharp fluctuations in temperature, humidity, light, sound
and ventilation should be avoided. The recommended space requirements for
animal rooms, for different species are given in (Annexure - 3).

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Anesthesia to Animals

Surgical and other procedures that cause pain or discomfort in animals must be performed under
general anesthesia. General anesthesia is also useful as a method of restraint for procedures that
if performed without anesthesia would cause excessive stress for the animal and/or expose the
human handler to potential hazards. In addition, because surgical and other invasive procedures
can be expected to cause pain or discomfort beyond the duration of general anesthesia pain relief
must be provided postoperatively.

Investigators are responsible for the assessment and management of all types of pain in their
research animals and must include a pain management plan in their research protocols.

Goals of anesthetic

The anesthetics and analgesics are selected to achieve the following:

• Provide precise anesthesia administration to assure that animals receive adequate anesthesia to
block pain sensation, to produce unconsciousness, and to produce immobility without creating life-
threatening anesthetic overdoses.

• Not interfere with the outcome of the study using the animal(s).

• Not result in excessive undesirable post-operative side effects.

• Not cause pain or distress on induction or recovery.

Inhalant Anesthesia
Inhalant anesthetics allow precise control of the depth and duration of anesthesia in research
animals and can be used in almost any species.

A variety of inhalant anesthetics are available, but isoflurane is the most commonly used
Injectable Anesthesia
List of drug with dose mentioned in below annexure.

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Euthanasia
Animal euthanasia (euthanasia from Greek: "good death") is the act of putting an animal to death or
allowing it to die by withholding extreme medical measures. Reasons for euthanasia include incurable
(and especially painful) conditions or diseases,[1] lack of resources to continue supporting the animal, or
laboratory test procedures. Euthanasia methods are designed to cause minimal pain and distress.

Reasons for euthanasia:

 Terminal illness, e.g. cancer or rabies

 Behavioral problems (usually ones that cannot be corrected) e.g. aggression - Canines that have
usually caused grievous bodily harm to either humans or other animals through mauling are usually
seized and euthanized ('destroyed' in British legal terms).
 Old age and deterioration leading to loss of major bodily functions, resulting in severe impairment of
the quality of life
 Lack of home or caretaker
 Research and testing – In the course of scientific research or testing, animals may be euthanized in
order to be dissected, to prevent suffering after testing, to prevent the spread of disease, or other
reasons.

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Physical Facilities

The physical condition and design of animal facility determine, to a great

extent, the efficiency and economy of this operation. The design and size of an
animal facility depend on the scope of institutional research activities, animals to be
housed, physical relationship to the rest of the institution, and geographic location. A
well planned, properly maintained facility is an important element in good animal
care.

LOCATION OF ANIMAL FACILITIES TO LABORATORIES

Good animal husbandry and human comfort and health protection require
physical separation of animal facilities from personnel areas such as offices, break
room, training and education room. Because…

 animals are very sensitive to their living conditions.

 not exposed to dust, smoke, noise, wild rodents, insects

and birds.

 the animal rooms should occupy about 50-60% of the total constructed
area and the remaining area should be utilized for services such as stores,
washing, office and staff, machine rooms, quarantine and corridors.

 The environment of animal room (Macro-Environment) and animal cage

(Microenvironment) are factors on which the production and experimental
efficiency of the animal depends. Since animals are very sensitive to
environmental changes, sharp fluctuations in temperature, humidity, light, sound
and ventilation should be avoided.

FUNCTIONAL AREAS:

 Specialized laboratories:
Or
 Individual areas contiguous with or near animal housing areas for such activities
as surgery, intensive care, necropsy, radiography, preparation of special diets,

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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experimental manipulation, treatment, and diagnostic laboratory procedures

containment facilities or
 Receiving and storage areas for food, bedding
 Pharmaceuticals and biologics, and supplies
 Space for administration, supervision, and direction of the facility
 Showers, sinks, lockers and toilets for personnel
 An area for washing and sterilization equipment and supplies,
 An autoclave for equipment
 Food, and bedding; and separate areas
 An area for repairing cages and equipment
 An area to store wastes prior to incineration or removal

PHYSICAL FACILITIES:
a) Building materials: efficient and hygienic, moisture-proof, fire-resistant, seamless
materials are most desirable for interior surfaces including vermin and pest
resistance.
b) Corridor(s) should be wide enough to facilitate the movement of personnel as
well as equipment and should be kept clean.
c) Utilities such as water lines, drain pipes, and electrical connections

ANIMAL ROOM DOORS

Doors should not be rust, vermin and dust proof. They should fit properly
within their frames and provided with an observation window.

EXTERIOR WINDOWS
Windows are not recommended for small animal facilities. However, where
power failures are frequent and backup power is not available, they may be necessary
to provide alternate source of light and ventilation. In primate rooms, windows can be
provided.
FLOORS
epoxy smooth, moisture proof, nonabsorbent, skid-proof, resistant to wear, acid, solvents,
adverse effects of detergents and disinfectants.

DRAINS
To prevent high humidity, drainage must be adequate to allow rapid
removal of water and drying of surfaces. At the inlet and outlets of the drains should
be fitted with wire mesh guard to prevent wild rodent entry.

STORAGE AREAS

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Separate storage areas should be designed for feed, bedding, cages and
materials not in use.
Refrigerated storage, separated from other cold storage, is essential for storage
of dead animals and animal tissue waste.

FACILITIES FOR SANITIZING EQUIPMENT AND SUPPLIES

An area for sanitizing cages and ancillary equipment is essential with adequate
water supply.

EXPERIMENTAL AREA

All experimental procedures in small animals should be carried out in a

separate area away from the place where animals are housed. Aseptic surgery for
large animals should include separate functional areas for surgical support, like a
preparation area, the operating theatre room or rooms, and an area for post-operative
& intensive care and for treatment of animals.

ENVIRONMENT
(a) Temperature and Humidity Control
Air conditioning is an effective means of regulating these environmental
parameters for laboratory animals.
Temp maintain between 18 to 29°C all times.
Humidity - 30% to 70%

(b) Ventilation
In renovating existing or in building new animal facilities, consideration
should be given to the ventilation of the animals' primary enclosures.
The animal facility and human occupancy areas should be ventilated separately.

(c) Power and Lighting

The electrical system should be safe and provide appropriate lighting.
A time-controlled lighting system should be used to ensure a regular diurnal
lighting cycle wherever required. Emergency power should be available in the event
of power failure.

(d) Noise Control

The facility should be provided with noise free environment.
Preferably less than 85 dB is desirable for rodents and non-human primates.

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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ANIMAL HUSBANDRY
(a) Caging Or Housing System:
most important elements to facilitate animal well being, meet research requirements, and
minimize experimental variables.
The housing system should:
 Provide space that is adequate, permit freedom of movement and normal
postural adjustments, and have a resting place appropriate to the species;
 Provide a comfortable environment
 Provide an escape proof enclosure that confines animal safety
 Provide easy access to food and water;
 Provide adequate ventilation
 Meet the biological needs of the animals, e.g., maintenance of body
temperature, urination, defecation, and reproduction;
 Keep the animals dry and clean, consistent with species requirements ;
 Facilitate research while maintaining good health of the animals.
They should be constructed of sturdy, durable materials and designed to
minimize cross-infection between adjoining units. Polypropylene, polycarbonate and
stainless steel cages should be used to house small lab animals.

(b) SOCIAL ENVIRONMENT

The social environment includes all interactions among individuals of a group
or among those able to communicate.
In selecting a suitable social environment, attention should be given whether
the animals are naturally territorial or communal and accordingly they should be
housed single or in groups.
In grouping animals, it is important to take into account population density
and ability to disperse; initial familiarity among animals; and age, sex, and social
rank.
Population density can affect reproduction, metabolism, immune responses,
and behavior. Group composition should be held as stable as possible, particularly for
canine, non-human primates, and other highly social mammals, because mixing of
groups or introducing new members can alter behavioral and physiological functions.
Non-human primates should have a run for free ranging activities:

(c) ACTIVITY
Provision should be made for animals with specialized locomotor pattern to
express their natural habitat, especially when the animals are held for long periods.
e.g., artificial trees, ropes, bars, and perches are appropriate for non-human primates.
Pens, runs, or other out-of-cage space provide more opportunity for exercise,
and their use is encouraged when holding dogs for long periods.
(d) FOOD

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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Animals should be fed with palatable, non-contaminated, and nutritionally

adequate food daily unless the experimental protocol requires otherwise.
Feeders should allow easy access, while avoiding contamination by urine and
feces.
Food should be provided in sufficient amounts to ensure normal growth in
immature animals and to maintain normal body weight, reproduction, and lactation in
adults.
Food should contain adequate nutrition, with proper formulation and
preparation; and ensure free from chemical and microbial contaminants; bioavailability of
nutrients should be at par with the nutritional requirements of the
animal. The animal feed should contain moisture, crude fibre, crude protein, essential
vitamins, minerals, crude fat and carbohydrate for providing appropriate nutrition.

Laboratory animal diets should not be manufactured or stored in facilities used

earlier for farm feeds or any products containing additives such as rodenticides,
insecticides, hormones, antibiotics, fumigants, or other potential toxicants.

Areas in which diets are processed or stored should be kept clean and enclosed
to prevent entry of insects or other animals.
Precautions should be taken if perishable items such as meats, fruits, and
vegetables are fed, because these are potential sources of microbiological and
chemical contamination and can also lead to variation in the amount of nutrients
consumed.
Diet should be free from heavy metals (e.g., Lead, Arsenic, Cadmium, Nickel,
Mercury), naturally occurring toxins and other contaminants.
Exposure to extremes of relative humidity, unsanitary conditions, light,
oxygen, and insects hasten the deterioration of food.

Meats, fruits, vegetables, and other perishable items should be refrigerated if

required to be stored. Unused, open food should be stored in vermin proof conditions
to minimize contamination and to avoid potential spread of disease causing agents.
Food hoppers should not be transferred from room to room unless cleaned and
properly sanitized.

(e) BEDDING
Bedding should be absorbent, free from toxic chemicals or other substances
that cause irritation, injure animals or personnel, and of a type not readily eaten by
animals. Bedding should be used in amounts sufficient to keep animals dry between
cage changes without coming into contact with watering tubes.
Bedding should be removed and replaced periodically with fresh materials as
often as necessary to keep the animals clean and dry.

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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In general it is ideal to change

the bedding twice a week or whenever requires.

Nesting materials for newly delivered pups should be provided wherever

needed (e.g. Paper cuttings, tissue paper, cotton etc.)

WATER
Animals should have continuous access to fresh, potable, uncontaminated
drinking water, according to their requirements. Periodic monitoring of microbial
contamination in water is necessary.
Watering devices, such as drinking nozzles and automatic waterers should be
examined routinely to ensure their proper operation. Sometimes it is necessary to train
animals to drink water from automatic watering devices.
It is better to replace fresh water bottles every day than to refill them,
however, if bottles are to be refilled, care should be taken that each bottle is replaced
on the cage properly from where it was removed.

SANITATION AND CLEANLINESS

Animal rooms,
corridors, storage spaces, and other areas should be properly cleaned with appropriate
detergents and disinfectants as often as necessary to keep them free of dirt, debris, and
harmful agents of contamination.
Cleaning utensils, such as mops, pails, and brooms, should not be transported
between animal rooms.
Cages should be sanitized before animals.
ater bottles, sipper nozzles stoppers, and other watering equipment should
be washed and then sanitized by rinsing with water of at least 82.2oC.

WASTE DISPOSAL
Wastes should be removed regularly and frequently. All waste should be
collected and disposed off in a safe and sanitary manner. The most preferred method
of waste disposal is incineration. Incinerators should be in compliance with all central,
state, and local Public Health and Pollution Control Board regulations.
Waste containers containing animal tissues, carcasses, and hazardous wastes
should be lined with leak - proof, disposable liner and dispose in bio waste disposable center.

PEST CONTROL
Adaptation of Programs designed to prevent, control, or eliminate the presence
of or infestations by pests are essential in an animal home environment.

EMERGENCY, WEEKEND AND HOLIDAY CARE

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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There should be an institutional policy to care animals by qualified personnel

every day, including weekends and holidays, to safeguards their well - being
including emergency veterinary care. In the event of an emergency, institutional
security personnel and fire or police officials should be able to reach responsible
persons for the animals.

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University

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RECORD KEEPING

It is essential that animal House should maintain following records:

1. Animal House plans, which includes typical floor plan, all fixtures etc.

2. Animal House staff record - both technical and non – technical

3. Health record of staff and animal

4. All SOPs relevant to experiments, care, breeding and management of animals

5. Breeding, stock, purchase and sales records

6. Minutes of institutional Animals Ethics Committee Meetings

7. Records of experiments conducted with the number of animals used (copy of

Form D)

8. Mortality, Postmortem Record

9. Clinical record of sick animals

10. Training record of staff involved in animal activities

11. Water, feed and bedding materials analysis report

12. Health monitoring Records

13. Rehabilitation Records

Dr. Pravin R. Tirgar, Professor, School of Pharmacy, RK University