Regulatory Framework in Ethics of Animal Research

Guidelines and Sop on the Regulation of Scientific Experiments on Animals

Introduction

1.1 Background

The scientific rationale for using animals in research was one of the concerns of the ethics committee initiated July 2005 – faculty of medicine – Alexandria University;

An initial guideline for ethics that should be followed in animal research was adopted at that time.

 The two principal questions which this frame work seeks to clarify are therefore:

■ does the scientific use of animals lead to valid, useful and relevant results in specific areas?

 ■ is it permissible for one species to cause pain, suffering and death to another to achieve aims that benefit primarily the human species?

Three main types of research can be distinguished.

 ■ Advancing scientific knowledge: i.e.  basic research, has no direct application and its primary purpose is to advance scientific knowledge about the way animals behave, or develop and function biologically. The study of basic physiological processes and genetic mechanisms also falls

 ■ Using animals as models for humans to study disease mechanisms and develop interventions. Animals are used as models for humans to understand disease processes and to develop effective preventative and therapeutic measures such as vaccines or medicines.

 ■ Animals as models in toxicity testing .Animals are used to test the safety of a range of compounds that are potentially hazardous for animals, humans or the environment.

Animal research is an essential part of many scientist work. Furthermore, certain provisions in the current regulatory framework for approval of chemical products and medicines require tests involving animals. We conclude that it is unrealistic to assume that all animal experimentation will end in the short term. It is crucial, therefore, to create a climate in which the necessity and justification for using animals is assessed and discussed fairly and with due respect for all views. Constructive debate would be facilitated by the provision of clear information about the full implications of research involving animals in terms of the kind, numbers and species of animals used, as well as the pain, suffering and distress to which they can be subjected.

It is equally important that society should be informed about the scientific, medical and other benefits of research involving animals. The freedom to promote or oppose research involving animals peacefully can be considered. Ethical issues as in using animals repeatedly in experiments for education or training is of importance here. Controversies about the acceptability of basic research therefore focus primarily on its usefulness and relevance, and on the ethical question of whether it is necessary and justifiable, if it causes specific degrees of pain, suffering or distress to the animals involved. The question of validity, usefulness and relevance is more complicated when animals are used as models for humans, as the question of whether reliable extrapolations can be made from one species to the other, needs to be addressed.

We conclude that because of evolutionary continuities in the form of behavioural, anatomical, physiological, neurological, biochemical and pharmacological similarities between animals and humans there are sufficient grounds for the scientific hypothesis that, in specific cases, animals can be useful models to study particular aspects of biological processes in humans, and to examine the effects of therapeutic and other interventions.

The Context of the debate

Statistical information about the number of animals used and the suffering involved The Annual Statistics of Scientific Procedures on Animals, published by the Home Office, have an important role in providing information about animal experimentation. At the same time, there is wide agreement that the data are presented in ways that are not readily accessible to lay people, and that the presentation could be improved. The Statistics have been criticized for not providing clear answers to the following questions:

 (i) What is the nature, level and duration of pain, suffering and distress experienced by animals used in the different kinds of procedures? And

 (ii) How many animals are used in procedures and related activities?

Balanced information: campaigning organizations

Detailed information about both the scientific benefits and the costs in terms of the implications for animal welfare. Similarly, the advantages and limitations of using alternative methods for research need to be discussed in a realistic manner.

1.2 Process of Evolution of the Guidelines

In 2017, the University of Alexandria, requests the presence of ethics committee for animal research - where the ethics committee took a decision to have a subcommittee concerned with animal research and formulate a regulation framework for animal research ethics instead of having a new committee.  The committee were followed AAALAC and modifications had been taken to suite the research environment in Egypt.  

Brainstorming session was held, wherein the principles and practices of utilization and care of animals in testing, research and training were finalized.

1.3 Aim

The aim of these Guidelines is to ensure ethical treatment of animals, while facilitating legitimate scientific research involving experiments on animals.

2. 2. Law of scientific experiments on animals

Until this moment, it is under discussion.

3.1 Ethical principles adopted by ethics committee for use of animals in scientific experiments

1- Experiments on animals carried out for the purposes of advancement by new discovery of knowledge which is expected to be useful for saving or prolonging human life or alleviating suffering; or for significant gains in the wellbeing for the people of the country; or for combating any disease, whether of human being, animals or plants.

2- Experiments should be designed with the minimum number of animals to give statistically valid results at 95% level of confidence.

3-If there are alternatives methods than animal testing, it should be given full consideration and sound justification.

4- Proper use of animals in experiments and avoidance or minimization (when avoidance is not possible) of pain and suffering inflicted on experimental animals should be an issue of priority for research personnel.

5- All scientific procedures adopted with animals that may cause more than momentary or slight pain and/or suffering should be performed with appropriate sedation, analgesia or anesthesia.

6- Persons engaged in animal experimentation have a moral responsibility for the welfare of the animals after their use in experiments.

7- Investigators are responsible for the aftercare and/or rehabilitation of animals after experimentation, and may be permitted to euthanize animals only in the following situations:

(a) When the animal is paralyzed and is not able to perform its natural functions.

(b) The animal has been left with a severe recurring pain and the animal exhibits obvious signs of long term extreme pain and suffering.

(c) Life threatening situations to human beings or other animals.

8- Costs of aftercare and/or rehabilitation of animal’s post-experimentation are to be part of research costs and should be scaled per animal in positive correlation with the level of sentience of the animals.

9- The living conditions of animals should be appropriate for their species and contribute to their health and comfort. The housing, feeding, and care of all.

10- Animals used for biomedical purposes must be directed by a veterinarian or other scientist in a relevant discipline who is trained and experienced in the proper care, handling, and use of the species being maintained or studied. In all circumstances, veterinary care shall be provided as necessary.

3.2.1 Guidelines­ on specific aspects regarding the use of animals in scientific experiments

3.2.1 Need to avoid/minimize pain and suffering inflicted on experimental animals

Proper use of animals in experiments and avoidance or minimization (when avoidance is not possible) of pain and suffering inflicted on experimental animals should be an issue of priority for research personnel, and unless the contrary is scientifically established, investigators should proceed on the basis that procedures that cause pain or suffering in human beings will also cause similar pain or suffering in animals. All scientific procedures adopted with animals that may cause more than momentary or slight pain and/or suffering should be performed with appropriate sedation, analgesia or anesthesia.

3.2.2 Proper care, handling and use of experimental animals

The living conditions of animals should be appropriate for their species and contribute to their health and comfort. The housing, feeding, and care of all animals used for biomedical purposes must be directed by a veterinarian or other scientist in a relevant discipline who is trained and experienced in the proper care, handling, and use of the species being maintained or studied. In all circumstances, veterinary care shall be provided as necessary.

3.2.3 Agricultural production research

The conventional regulatory framework can be modified to suit the use of experimental animals in agricultural production research.

3.2.4 Powers of the Animals Ethics Sub- Committee (AESC)

AESC is empowered to clear research project proposals that involve experiments on animals in the medical field.

3.2.5 Inspection of animal house facilities

Both announced and unannounced visits by duly authorized personnel (only) to inspect the animal house facilities of institutes may be carried out.

4. 4. Procedures for approval of scientific experiments on animals

4.1 Definition of experiment:

“Experiments” means any programme or project involving use of animal(s) for the acquisition of knowledge of a biological, physiological, ethological, physical or chemical nature; and includes the use of animals(s) in the production of reagents and products such as antigens and antibodies, routine diagnostics, testing activity and establishment of transgenic stocks, for saving or prolonging life or  alleviating suffering, or significant gains in the well-being for people of the country or for combating any disease, whether of human beings, animals or plants.

4.2 Experimental animals which are subject to regulation, the relative sentience of different species of animals are as follows:

Invertebrates (e.g., cockroaches) <Birds <Rodents <Canines/Felines <Bovine/Equines <Primates (e.g., Rhesus Macaque) <More evolved Primates (e.g., chimpanzee) anything higher than invertebrates in terms of level of sentience requires regulation. Thus rats, mice, birds, and farm animals are also subject to regulation.

4.3 Function of Animals Ethics Sub- Committee (AESC)

All establishments engaged in research and education involving animals, are required to comply with the various guidelines, norms and stipulations set out by (AESC).

The main functions of (AESC) are:

1. Registration of establishments conducting animal experimentation or breeding of animals for this purpose.
2. Selection and appointment of nominees in the subcommittee
3. Ethics Committees of registered establishments.
4. Approval of Animal House Facilities based on reports of inspections conducted by (AESC).
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.

4.4 Constitution of the (AESC)

It includes two scientists or more from different biological disciplines, in addition to a pharmacologist, surgeon and the lay person in the Ethics committee should attend the meeting. A specialist may be co-opted while reviewing special projects using hazardous agents such as radioactive substances and deadly microorganisms.

A veterinarian can be consulted in certain situations.

4.5 Approval of animal house facilities

Approval of animal house facilities by (AESC) is required to be obtained, for premises where experiments are to be conducted.

4.6 Use of animals in experiments

 animals lowest on the phylogenetic scale which may give scientifically valid results should be first considered for any experimental procedure, and the experiment should be designed with the minimum number of animals to give statistically valid results at 95% degree of confidence.

4.7 Procurement of animals

 (i) An establishment shall acquire animals for experiments from registered breeders only;

(ii) In case of non-availability of animals from registered breeders, the animals may be procured from alternative legal sources;

4.8 Welfare of animals during use in experiments

Personnel using the experimental animals shall be responsible for the welfare of the animals during their use in experiments.

4.9. Aftercare and rehabilitation of animals after use in scientific experiments

Investigators shall be responsible for the aftercare and rehabilitation of the animals after experimentation.

Rehabilitation treatment of an animal after experimentation shall extend till

4.10 Situations Where euthanasian of animals is permissible

Investigators shall not euthanize animals except in situations as defined below:

• When the animal is paralyzed and is not able to perform its natural functions or it becomes incapable of independent locomotion or it can no longer perceive the environment in an intelligible manner; or
• If during the course of experimental procedure the animal has been left

with a recurring pain wherein the animal exhibits obvious signs of pain

and suffering; or

(iii) Where the non-termination of the life of the experimental animal will be life threatening to human beings or other animals.

4.11 Suspension/revocation of registration of an establishment by (AESC)

 (i) the rule made by (AESC) are not being complied with by an establishment or breeder; or

(ii) a violation of the directions of the (AESC)  has been committed by any establishment or breeder and the Committee’s directions to rectify such violation has not been complied within the period so specified, Provided further that no order for suspension or revocation of registration, or closure of animal house facility shall be issued in a case of minor violation. Explanation: For this clause, “minor violation” means an act of commission or omission which does not have direct bearing on the health of an animal; which may not lead to adverse health effect or pain or suffering or death of an animal.

(AESC) GUIDELINES FOR LABORATORY ANIMAL FACILITY

Good Laboratory Practices (GLP) for animal facilities is intended to assure quality maintenance and welfare of animals used in laboratory studies while conducting biomedical and behavioral research and testing of products.

GOAL

The goal of these Guidelines is to promote the humane care of animals used in biomedical and behavioral research and testing with the basic objective of providing specifications that will enhance animal wellbeing, quality in the pursuit of advancement of biological knowledge that is relevant to humans and animals.

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.
• Daily observation of animals can be accomplished by someone other than a veterinarian; however, a mechanism of direct and frequent communication should be adopted so that timely and accurate information on problems in animal health, behavior, and wellbeing is conveyed to the attending veterinarian.

ANIMAL PROCUREMENT

• All animals must be acquired lawfully as per the (AESC) guidelines.
• A health surveillance program for screening incoming animals should be carried out to assess animal quality. Methods of transportation should also be taken into account.
• The animals should be quarantined and stabilized according to procedures appropriate for the species and circumstances.

QUARANTINE, STABILIZATION AND SEPARATION

• Quarantine is the separation of newly received animals from those already inthe 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, psychologic 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 required and after handling these animals they should not be handling any other animals in the facilities

SURVEILLANCE, DIAGNOSIS, TREATMENT AND CONTROL OFDISEASE

• 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.
• 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.

ANIMAL CARE AND TECHNICAL PERSONNEL

• Animal care programs require technical 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 program

PERSONAL HYGIENE

• 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 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.

ANIMAL EXPERIMENTATION INVOLVING HAZARDOUS AGENTS

The use of animals in such studies requires special considerations, the procedures and the facilities to be used must be reviewed by both the Institutional Bio-safety committee and (AESC).

MULTIPLE SURGICAL PROCEDURES ON SINGLE ANIMAL

Multiple surgical procedures on a single animal for any testing or experiment are not to be practiced unless specified in a protocol only approved by the (AESC).

DURATIONS OF EXPERIMENTS

No animal should be used for experimentation for more than 3 years unless adequate justification is provided.

PHYSICAL RESTRAINT

• Brief physical restraint of animals for examination, collection of samples, and a variety of other clinical and experimental manipulations can be accomplished manually or with devices be suitable in size and design for the animal being held and operated properly to minimize stress and avoid injury to the animal.
• Prolonged restraint of any animal, including the chairing of non-human primates, should be avoided unless essential to research objectives.
• Less restrictive systems, such as the tether system or the pole and collar system should be used when compatible with research objectives.

The following are i­­mportant guidelines for the use of restraint equipment’s:

• Restraint devices cannot be used simply as a convenience in handling or managing animals.
• The period of restraint should be the minimum required to accomplish the research objectives.
• Animals to be placed in restraint devices should be given training to adapt to the equipment.
• Observation of the animal is mandatory.
• Special care should be provided if lesions or illness associated with restraint are observed.
• The presence of lesions, illness, or severe behavioral change should be dealt with by the temporary or permanent removal of the animal from restraint.

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 depends 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

Separation of animal facilities from personnel areas such as offices, break room, training and education room is mandatory:

• 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.
• 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.
• Sharp fluctuations in temperature, humidity, light, sound and ventilation should be avoided.

FUNCTIONAL AREAS

The size and nature of a facility will determine whether areas for separate service functions are possible or necessary. Sufficient animal area required to:

• Ensure separation of species or isolation of individual projects when necessary;
• Receive, quarantine, and isolate animals; and
• Provide for animal housing.
• 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, experimental manipulation, treatment, and diagnostic laboratory procedures containment facilities or Equipment, if hazardous biological, physical, or chemical agents are to be used
• 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
• For holding soiled and cleaned equipment
• An area for repairing cages and equipment
• An area to store wastes prior to incineration or removal

PHYSICAL FACILITIES

• (a) Building materials should be selected to facilitate efficient and hygienic operation of animal facilities. Durable, 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 should preferably be accessible through service panels or shafts in corridors outside the animal rooms.

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. Door closures may also, be provided. Rodent barriers can be provided in the doors of the small animal facilities.

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

Floors should be either monolithic or epoxy smooth, moisture proof, nonabsorbent, skid-proof, resistant to wear, acid, solvents, adverse effects of detergents and disinfectants. They should be capable of supporting racks, equipment, and stored items without becoming gouged, cracked, or pitted, with minimum number of joints.

A continuous moisture-proof membrane might be needed. If sills are installed at the entrance to a room, they should be designed to allow for convenient passage of equipment.

DRAINS

Floor drains are not essential in all rooms used exclusively for housing rodents. Floor in such rooms can be maintained satisfactorily by wet vacuuming or mopping with appropriate disinfectants or cleaning compounds. Where floor drains are used, the floors should be sloped and drain taps kept filled with water or corrosion free mesh. 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

WALLS & CEILINGS

Walls should be free of cracks, unsealed utility penetrations, or imperfect junctions with doors, ceilings, floors and corners.

Surface materials should be capable of withstanding scrubbing with detergents, disinfectants and the impact of water under high pressure.

STORAGE AREAS

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. Temperature and humidity control prevents variations due to changing climatic conditions keeping in view of the variations in the number of room occupants the range should be within or approximately between 18 to

29°C (64.4 to 84.2oF) all times.

The relative humidity should be under control within the range of 30% to 70% throughout the year. For larger animals a comfortable zone (18 to 37°C) should be maintained. During extreme summer appropriate methods e.g. sprinklers should be adopted for cooling.

(b) Ventilation

In renovating existing or in building new animal facilities, consideration should be given to the ventilation of the animals' primary enclosures. Heating, ventilation, and air-conditioning systems should be designed with 12-

15 air cycles per hour so that operation can be continued with a standby system. 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 and with sufficient number of power points lighting system be installed provide adequate illumination for people to work in the animal rooms and a lowered intensity of light for the animals.

Fluorescent lights are efficient and less than 400 lux is preferable for rodent facilities.

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. Noise control is an important consideration in designing the animal facility. Concrete walls are more effective than metal or plaster walls because their density reduces sound transmission. Preferably less than 85 dB is desirable for rodents and non-human primates. 30

ANIMAL habitant

(a) Caging or Housing System

The caging or housing system is one of the most important elements in the physical and social environment of research animals. It should be designed carefully to facilitate animal wellbeing, 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, Monkeys should be housed in cages made of steel or painted mild steel and for other animals such as sheep, horses.

To simplify servicing and sanitation, cages should have smooth, impervious surfaces that neither attract nor retain dirt and a minimum number of ledges, angles, and corners in which dirt or water can accumulate.

The design should allow inspection of cage occupants without disturbing them. Feeding and watering devices should be easily accessible for filling, changing, cleaning and servicing. Cages, runs and pens must be kept in good condition to prevent injuries to animals, promote physical comfort, and facilitate sanitation and servicing. Particular attention must be given to eliminate sharp edges and broken wires, keeping cage floors in good condition.

SHELTERED OR OUTDOOR HOUSING

• When animals are maintained in outdoor runs, pens, or other large enclosures, there must be protection from extremes in temperature or other harsh weather conditions an adequate protective and escape mechanism for submissive animals especially in monkeys by way of providing indoor portion of run.
• Shelter should be accessible to all animals, with sufficient ventilation, and should be designed to prevent accumulation of waste materials and excessive moisture.
• Houses, dens, boxes, shelves, perches, and other furnishings should be constructed in a manner and made of materials that allow cleaning or replacement in accordance with generally accepted husbandry practices when the furnishings are soiled or worn out.
• Ground-level surfaces of outdoor housing facilities can be covered with absorbent bedding, sand, gravel, grass, or similar material that can be removed or replaced when that is needed to ensure appropriate animation.
• Accumulation of animal waste and stagnant water should be avoided by, for example, using contoured or drained surface. Other surfaces should be able to withstand the elements and be easily maintained.

 

SOCIAL ENVIRONMENT

• The social environment includes all interactions among individuals of a group or among those able to communicate. The effects of social environment in caged animals vary with the species.
• 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.
• When appropriate, group housing should be considered for communal animals. In grouping animals, it is important to consider 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:

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.
• Cages are often used for short-term (up to 3 months) housing of dogs and may be necessary for postsurgical care, isolation of sick dogs, and metabolic studies.
• Pens, runs, or other out-of-cage space provide more opportunity for exercise, and their use is encouraged when holding dogs for long periods.

FOOD

• 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.

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 meeting watering tubes.
• Bedding should be removed and replaced periodically with fresh materials as often as necessary to keep the animals clean and dry. The frequency is a matter of professional judgment of animal care personnel in consultation with the investigation depending on the number of animals and size of cages. In general, it is ideal to change the bedding twice a week or whenever requires.
• The desirable criteria for rodent contact bedding is ammonia binding, serializable, deleterious products not formed as a result of sterilization, easily stored, non - desiccating to the animal, uncontaminated, unlikely to be chewed or mouthed, non - toxic, non - malodorous, nest able, disposable by incineration, readily available, remains stable during use, manifests batch uniformity, optimizes normal animal behavior, non - deleterious to cage - washers, non - injurious and non - hazardous to personnel, non - nutritious and non - palatable. 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

• Sanitation is an essential activity in an animal facility. 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.
• Where animal waste is removed by hosing or flushing, this should be done at least twice a day. Animals should be kept dry during such procedures. For larger animals, such as dogs, cats, and non - human primates, soiled litter material should be removed twice daily.
• Cages should be sanitized before animals are placed in them. Animal cages, racks, and accessory equipment’s, such as feeders and watering devices, should be washed and sanitized frequently to keep them clean and contamination free.
• Generally, this can be achieved by washing solid bottom rodent cages and accessories once or twice a week and cages, racks at least monthly.

Wire - bottom cages other than rodent cages should be washed at least every 2 weeks. It is good practice to have extra cages available always so that a systematic cage-washing schedule can be maintained. Cages can be disinfected by rinsing at a temperature of 82.2C (180oF) or higher for a period long enough to ensure the destruction of vegetative pathogenic organisms.

• Disinfection can also be accomplished with appropriate chemicals.
• Equipments should be rinsed free of chemicals prior to use. Periodic microbiologic monitoring is useful to determine the efficacy of disinfection or sterilization procedures.
• Rabbits and some rodents, such as guinea pigs, mice and hamsters, produce urine with high concentration of proteins ammonia and minerals. Minerals and organic compounds in the urine from these animals often adhere to cage surfaces and necessitate treatment with acid solutions before washing.
• Water bottles, sipper nozzles stoppers, and other watering equipment should be washed and then sanitized by rinsing with water of at least 82.2oC (180oF) or appropriated chemicals agents (e.g. Sodium Hyperchlorite) to destroy pathogenic organisms, if bottles are washed by hand, mechanized brushes at the washing sink are useful, and provision should be made for dipping or soaking the water bottles in detergents and disinfectant solutions. A two – compartment sink or tub is adequate for this purpose.
• Some means for sterilizing equipment's and supplies, such as an autoclave or gas sterilizer, is essential when pathogenic organisms are present. Routine sterilization of cages, feed and bedding is also essential besides care is taken to use clean materials from reliable sources. Where hazardous biological, chemical, or physical agents are used, a system of equipment monitoring might be appropriate.
• Deodorants or chemical agents other than germicidal agents should not be used to mask animal odors. Such products are not a substitute for good sanitation.

ASSESSING THE EFFECTIVENESS OF SANITATION

• Sanitation practices should be monitored appropriately to ensure effectiveness of the process and materials being cleaned; it can include visual inspection of the materials, monitoring of water temperatures, or microbiologic monitoring.
• The intensity of animal odors particularly that of ammonia should not be used as the sole means of assessing the effectiveness of the sanitation program.
• A decision to change the frequency of such bedding changes or cage washing should be based on factors such as the concentration of ammonia, appearance of the cage, condition of the bedding and number and size of the animals housed in the cage.

Autoclaving: Chemical Indicator - batch wise assessment; Biological indicator - Periodical assessment.

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 follow 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 liners. If wastes must be stored before removal, the waste storage area should be separated from other storage facilities and free of flies, cockroaches, rodents, and other vermin. Cold storage might be necessary to prevent decomposition of biological wastes. Hazardous wastes should be rendered safe by sterilization, decontamination, or other appropriate means before they are disposed off from an animal facility.

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

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. That can be enhanced by prominently posting emergency procedures, names, or telephone numbers in animal’s facilities or by placing them in the security department or telephone center. A disaster plan that considers both personnel and animals should be prepared as part of the overall safety plan for the animal facility.

RECORD KEEPING

It is essential that animal House should maintain following records:

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

􀂾 Animal House staff record - both technical and non - technical

􀂾 Health record of staff and animals

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

􀂾 Breeding, stock, purchase and sales records

􀂾 Minutes of institutional Animals Ethics Committee Meetings

􀂾 Records of experiments conducted with the number of animals used (copy of Form D)

􀂾 Mortality, Postmortem Record

􀂾 Clinical record of sick animals

􀂾 Training record of staff involved in animal activities

􀂾 Water, feed and bedding materials analysis report

􀂾 Health monitoring Records

􀂾 Rehabilitation Records

STANDARD OPERATING PROCEDURES (SOPs) / Guidelines

The Institute should maintain SOPs describing procedures / methods adapted about Animal Husbandry, maintenance, breeding, animal house activities microbial testing and experimentation.

A SOP should contain the following items:

􀂾 Name of the Author

􀂾 Title of the SOP

􀂾 Date of approval

􀂾 Reference of previous SOP on the same subject and date (Issue no and Date)

􀂾 Location and distribution of SOP’s with sign of each recipient 37

􀂾 Objectives

􀂾 Detailed information of the instruments used in relation with animals with methodology (Model no., Serial no., Date of commissioning, etc)

􀂾 The name of the manufacturer of the reagents and the methodology of the analysis pertaining to animals

􀂾 Normal value of all parameters

􀂾 Hazard identification and risk assessment

PERSONNEL AND TRAINING

The selection of animal facility staff, particularly the staff working in animal rooms or involved in transportation, is a critical component in the management of an animal facility.

The staff must be provided with all required protective clothing (face masks, head covers, aprons, gloves, gumboots, other footwear etc.) while working in animal rooms. Facilities should be provided for change over with lockers, wash basin, toilets and bathrooms to maintain personal hygiene. It is also important a regular medical check-up is arranged for the workers to ensure that they have not picked up any zoonotic infection and also that they are not acting as a source of transmission of infection to the animals. The animal house in-charge should ensure that persons working in animal house don’t eat, drink, smoke in animal room and have all required vaccination, particularly against Tetanus and other zoonotic diseases.

Initial in-house training of staff at all levels is essential. A few weeks must be spent on the training of the newly recruited staff, teaching them the animal handling techniques, cleaning of cages and importance of hygiene, disinfection and sterilization. They should also be made familiar with the activities of normal healthy and sick animals so that they are able to spot the sick animal during their daily routine check up of cages.

TRANSPORT OF LABORATORY ANIMALS

The transport of animals from one place to another is very important and must be undertaken with care. The main considerations for transport of animals are, mode of transport, containers, animal density in cages, food and water during transit, protection from transit infections, injuries and stress.

The mode of transport of animals depends on the distance, seasonal and climatic conditions and the species of animals. Animals can be transported by road, rail or air taking into consideration of above factors. In any case the transport stress should be avoided and the containers should be of an appropriate size so as to enable these animals to have a comfortable, free movement and protection from possible injuries. The food and water should be provided in suitable containers or in suitable form so as to ensure that they get adequate food and more particularly water during transit. The transport containers (cages or crates) should be of appropriate size and 38 only a permissible number of animals should only be accommodated in each container to avoid overcrowding and infighting

ANAESTHESIA AND EUTHANASIA

The investigators should ensure that the procedures, which are considered painful, are conducted under appropriate anaesthesia as recommended for each species of animals.

It must also be ensured that the anaesthesia is given for the full duration of experiment and at no stage the animal is conscious to perceive pain during the procedure. If at any stage during the experiment the investigator feels that he has to abandon the experiment or he has inflicted irreparable injury, the animal should be humanely sacrificed. Neuromuscular blocking agents must not be used without adequate general anaesthesia).

In the event of a decision to sacrifice an animal or termination of an experiment or otherwise an approved method of euthanasia should be adopted and the investigator must ensure that the animal is clinically dead before it is sent for disposal. The data of all the animals, that have been euthanised, should be maintained.

Anaesthesia

Unless contrary to the achievement of the results of study, sedatives, analgesics and anaesthetics should be used to control pain or distress under experiment. Anaesthetics Agents generally affect cardiovascular, respiratory and thermo-regulatory mechanism in addition to central nervous system.

Before using actual anaesthetics the animals are prepared for anaesthesia by overnight fasting and using pre-anaesthetics, which block parasympathetic stimulation of cardio-pulmonary system and reduce salivary secretion. Atropine is most commonly used anti-cholinergic agent. Local or general anaesthesia may be

used, depending on the type of surgical procedure.

Local anaesthetics are used to block the nerve supply to a limited area and are used only for minor and rapid procedures. This should be carried out under an expert supervision for regional infiltration of surgical site, nerve blocks and for epidural and spinal anaesthesia.

A number of general anaesthetic agents are used in the form of inhalants.

General anaesthetics are also used in the form of intravenous or intra-muscular injections such as barbiturates. Species characteristics and variation must be kept in mind while using an anaesthetic. Side-effects such as excess salivation, convulsions, excitement and disorientation should be suitably prevented and controlled. The

animal should remain under veterinary care till it completely recovers from anaesthesia and postoperative stress.

Euthana­sia

Euthanasia is resorted to events where an animal is required to be sacrificed or termination of an experiment or otherwise for ethical reasons. The procedure should be carried out quickly and painlessly in an atmosphere free from fear or anxiety. For accepting an euthanasia method as humane it should have an initial depressive action

on the central nervous system for immediate insensitivity to pain. The choice of a method will depend on the nature of study, the species of animal to be killed.

. The method should in all cases meet the following requirements:

(a) Death, without causing anxiety, pain or distress with minimum time lag phase.

(b) Minimum physiological and psychological disturbances.

(c) Compatibility with the purpose of study and minimum emotional effect on the operator.

(d) Location should be separate from animal rooms and free from environmental contaminants.

Tranquilizers have to be administered to larger species such as monkeys, dogs and cats before an euthanasia procedure.

LABORATORY ANIMAL ETHICS

All scientists working with laboratory animals must have a deep ethical consideration for the animals they are dealing with. From the ethical point of view it is important that such considerations are taken care at the individual level, at institutional level and finally at the national level.

TRANSGENIC ANIMALS

Transgenic animals are those animals, into whose germ line foreign gene(s) have been engineered, whereas knockout animals are those whose specific gene(s) have been disrupted leading to loss of function. These animals can be bred to establish transgenic animal strains. Transgenic animals are used to study the biological functions of specific genes, to develop animal models for diseases of humans or animals, to produce therapeutic products, vaccines and for biological screening, etc. These can be either developed in the laboratory or produced for R&D purpose from registered scientific/academic institutions or commercial firms, and generally from abroad with approval from appropriate authorities.

MAINTENANCE

Housing, feeding, ventilation, lighting, sanitation and routine management practices for such animals are similar to those for the other animals of the species as given in guidelines. However, special care has to be taken with transgenic/gene knockout animals where the animals can become susceptible to diseases where special conditions of maintenance are required due to the altered metabolic activities. The transgenic and knockout animals carry additional genes or lack genes compared to the wild population. To avoid the spread of the genes in wild population care should be taken to ensure that these are not inadvertently released in the wild to prevent cross breeding with other animals. The transgenic and knockout animals should be maintained in clean room environment or in animal isolators.

DISPOSAL

The transgenic and knockout animals should be first euthanized and then disposed off as described elsewhere in the guidelines. A record of disposal and the manner of disposal should be kept as a matter of routine.

BREEDING AND GENETICS

For initiating a colony, the breeding stock must be procured from ASCE registered breeders or suppliers ensuring that genetic makeup and health status of animal is known. In case of an inbred strain, the characters of the strain with their gene distribution and the number of inbred generation must be known for further propagation. The health status should indicate their origin, e.g. conventional, specific pathogen free or transgenic, gnotobiotic or knockout stock.