Maintenance of Laboratory Animals as per CPCSEA Guidelines

All establishments engaged in research and education involving animals in India are required to comply with the various guidelines, norms and stipulations set out by CPCSEA. The aim of these guidelines is to ensure humane and ethical treatment of animals, while facilitating legitimate scientific research involving experiments on animals and to make judicious use of animals for experimental purposes.

Committee for Purpose of Control andSupervision of Experiments on Animals (CPCSEA):

The Prevention of Cruelty to Animals Act 1960 as amended in 1982, is to prevent the 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 ensures 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.

Mandate of CPCSEA:

1. Registration of establishments conducting experiments on animals.
2. Registration of establishments engaged in breeding of laboratory animals.
3. Constitution of IAECs in the establishments registered with CPCSEA.
4. Approval of animal house facilities for small and large animals.
5. Permission for conducting experiments on large animals.
6. Recommendation for import of animals for experimentations and breeding.

CPCSEA has issued the following guidelines:

1. Guidelines for the Reuse and Rehabilitation of dogs.
2. Guidelines for Constitution/Reconstitutions of IAECs.
3. Care and Management of Equines used in the product of biologicals.
4. Standard operating procedures for IAEC.

Maintenance of laboratory animals as per CPCSEA guidelines:

CPCSEA has set guidelines for laboratory animal facility. 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 well being, quality in the pursuit of advancement of biological knowledge that is relevant to humans and animals. The CPCSEA provides the guidelines to be followed by all research institutions in the country.

1. Adequate veterinary care must be provided by a veterinarian or a person who has training or experience in laboratory animal sciences and medicine. Institutions should employ people trained in laboratory animal science or provide for both formal and on-the-job training for the care of animals. It is essential that the animal care staff maintain a high standard of personal cleanliness.
2. All animals must be acquired lawfully as per the CPCSEA guidelines. 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 the mode of transport, the containers, the animal density in cages, food and water during transit, protection from transit infections, injuries and stress. Quarantine is the separation of newly received animals from those already in the facility. An effective quarantine minimizes the chance for introduction of pathogens into an established colony.
3. All animals should be observed for signs of illness, injury, or abnormal behavior. Health and nutritional status of the animals should be properly maintained.
4. Animals should be fed palatable, non-contaminated and nutritionally adequate food daily unless the experimental protocol requires otherwise. Ordinarily animals should have continuous access to fresh, potable, uncontaminated drinking water, according to their particular requirements. 
5. 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 location, building, cages, material and environment of animal rooms are the major factors, which affect the quality of animals.
6. Housing, care, breeding and maintenance of experimental animals is necessary to keep them in physical comfort, good health and behave normally. Bedding should be removed and replaced with fresh materials as often as necessary to keep the animals clean and dry.
7. Sanitation is essential in an animal facility. Animal rooms, corridors, storage spaces, and other areas should be cleaned with appropriate detergents and disinfectants as often as necessary.
8. The Institute shall maintain SOPs describing procedures/methods adapted with regard to animal husbandry, maintenance, breeding, animal house microbial analysis and experimentation records. The animal house should maintain the different records of animals as instructed by CPCSEA.
9. Acceptable experimental techniques and procedures for anaesthesia and euthanasia are applied during the study.
10. 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. 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.
11. 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.

Alternatives to animal screening procedures: 3 Rs concept

British researchers William Russell and Rex Burch in 1959 formulated this concept of the 3 Rs in their book ‘The Principles of Humane Experimental Technique’, which argues that humane science is the best science. Alternative methods fall into three broad categories. These are called the 3 Rs: Replacement, Reduction, and Refinement. Since the introduction of these principles, they have become widely accepted internationally as the basic principles guiding animal use in research, teaching and testing. Ron Banks added the 4^th R i.e. Rehabilitation and Reuse of animals.

Institutional Animal Ethics Committee (IAEC):

As defined in “Breeding of and Experiments on Animals (Control and Supervision) Rules, 1998”: Institutional Animals Ethics Committee means a body comprising of a group of persons recognized and registered by the CPCSEA performed in an establishment which is constituted and operated in accordance with procedures specified for the purpose by the committee. The primary responsibility of a person who has been nominated to represent CPCSEA on IAEC is the wellbeing and welfare of the animals housed or kept for experiments/breeding.

Composition and Constitution of IAEC

IAEC includes 8 members as described below:

A. Institutional Members (5 members from the institute)

1. A Scientist In Charge of Animals Facility
2. A Veterinarian Involved In the Care of Animals
3. A Biological Scientist
4. Two Scientists from Different Biological Disciplines

B. CPCSEA Nominated members (3+1 members from outside institute)

1. A CPCSEA Main Nominee
2. A CPCSEA Link Nominee
3. A Scientist From Outside the Institute
4. A Socially Aware Nominee

The Chairperson and member secretary of the committee are nominated by the institution and should take care of the functioning of IAEC in accordance with procedures specified for the purpose by the committee.

Objectives of IAEC:

1. Experiments to be avoided wherever it is possible to do so.
2. Experiments on larger animals are avoided when same results can be achieved in smaller animals.
3. Rationalize usage of animals- 3 Rs.
4. Animals to be properly looked after before and after experiments.

The main functions of IAEC are:

1. IAEC will review of research proposals and prevent infliction of unnecessary pain and sufferings before, during and after experiments on animals, to follow the CPCSEA guidelines.
2. IAEC reviews and approves all research proposals involving small laboratory animal experiments with a view to assure quality maintenance and welfare of animals used in pre-clinical research.
3. For experiments on large animals, the IAEC will forward its recommendation to the CPCSEA, for its approval process.
4. IAEC ensures that experiments shall be performed in every case by or under the supervision of a qualified person and under the responsibility of the Principle Investigator.
5. IAEC reviews the proposals before start of the study as well as monitor the research throughout the study and after completion of the study through annual reports, final report.
6. IAEC team will visit the laboratory in the animal house/respective department where the experiments are conducted. The committee also ensures compliance with all regulatory requirements, applicable guidelines and laws.
7. Monitor and inspect the housing of animals and ensure that it is as per specified standards.

Institutional Biosafety Committee (IBSC):

The Institutional Biosafety Committee shall be the point for interaction within institution for implementation of the guidelines. Any research project which is likely to have biohazard potential (as envisaged by the guidelines) during the execution stage or which involve the production of either micro-organisms or biologically active molecules that might cause biohazard should be notified to IBSC. IBSC will allow genetic engineering activity on classified organisms only at places where such work should be performed as per guidelines. Provision of suitable safe storage facility of donor, vectors, recipients and other materials involved in experimental work should be made and may be subjected to inspection on accountability.

The Institutional Biosafety Committee is engaged in hazardous chemical use, genetic engineering research and production activities. This committee shall also examine the proposal on animal experiments involving hazardous agents in addition to its existing functions. IBSC whose members are knowledgeable about hazardous agents, are in place in most of the higher level education, research institutes and in many pharmaceutical industries for safety issues. Institutional Biosafety Committee (IBSC) is to be constituted in all centres engaged in genetic engineering research and production activities. The committee will constitute the following.

1. Head of the institute or his nominee

2. Three or more scientist engaged in DNA work or molecular biology with an outside expert in the relevant discipline.

3. A member with medical qualification- Biosafety officer (in case of work with pathogenic agents/ large scale used).

4. One member nominated by Department of Biotechnology (DBT), Government of India.

The main functions of IBSC

The functions and activities of IBSE include the following:

1. Registration of Biosafety Committee membership composition with Review Committee on Genetic Manipulation (RCGM) and submission of report. ISBC will provide half yearly reports on the ongoing projects to RCGM regarding the observance of the safety guidelines on accidents, risks and on deviations if any. A computerized Central Registry for collation of periodic reports on approved projects will be setup with RCGM to monitor compliance on safeguards as stipulated in the guidelines.

2. Review and clearance of project proposals falling under restricted category that meets the requirements under the guidelines. IBSC would make efforts to issue clearance certificates quickly on receiving the research proposals from investigators.

3. Tailoring biosafety program to the level of risk assessment.

4. Training of personnel on biosafety.

5. Instituting health monitoring program for laboratory personnel, complete medical check-up of personnel working in projects involving work with potentially dangerous microorganism should be done prior to starting such projects. Follow up medical check-up including pathological test should be done periodically, annually for scientific workers involved in such projects. Their medical record should be accessible to the RCGM. It will provide half yearly reports on the ongoing projects to RCGM regarding the observance of the safety guidelines on accidents, risks and on deviations if any.

6. Adopting emergency plans.

Note: Above mentioned mandate and guidelines are documented in the CPCSEA websites i.e. http://cpcsea.nic.in.

Other guidelines for maintenance of laboratory animals

The Organization for Economic Cooperation and Development (OECD) and the International Conference on Harmonization (ICH) are the different organizations that have issued certain guidelines regarding toxicity and safety studies in animals.

The OECD has issued a guideline for toxicity testing of various chemicals. OECD is an intergovernmental organization in which representatives of 29 industrialized countries in North America, Europe and the Pacific. The OECD Secretariat located in Paris, France and its work is conducted by various committees and subsidiary groups. The work of the OECD related to chemical safety is carried out in the Environment, Health and Safety program. The OECD has issued the OECD test guidelines for chemical testing i.e. collection of methods used to assess the hazards of chemicals and of chemical preparations such as pesticides. These methods cover tests for physical and chemical properties, effects on human health and wild life, accumulation and degradation in the environment. The OECD provides the guidelines on selection of animal species, housing and feeding conditions and preparation of animals for toxicity study. OECD test Guideline 420, 423 and 425 are used for acute toxicity study and 452 for chronic toxicity study. The OECD test guidelines are recognized worldwide as the standard reference tool for chemical testing.

The ICH is a sole project that unites the regulatory authorities of Europe, Japan and the United States.  The experts from pharmaceutical industries from the above three regions should discuss scientific and technical aspects of product registration. The object of such harmonization is a more economical use of human, animal and material resources and the elimination of unnecessary delay in the global development and availability of new medicines by maintaining the quality, safety and efficacy and regulatory obligations to protect public health. The ICH guidelines are divided into four major categories i.e. a) Quality: relating to chemical and pharmaceutical quality assurance, b) Safety- relating to in vitro and in vivo preclinical studies, c) Efficacy- relating to clinical studies, and d) Multidisciplinary- topics that do not fit uniquely into one of the above categories.

Note: Certain guidelines regarding toxicity and safety studies in animals for new chemical or pharmaceutical products are placed in both OECD and ICH. These guidelines are documented in the websites of WWW.oecd.org and WWW.ich.org.

References:

Committee for Purpose of Control and Supervision of Experiments on Animals, 2017. General Guidelines for Nominees of CPCSEA. Government of India, Ministry of  Environment, Forest and Climate Change, Animal welfare Division., CPCSEA, New Delhi, India.

Committee for the Purpose of Control and Supervision on Experiments on Animals, 2003. CPCSEA Guidelines for Laboratory Animal Facility. Indian Journal of Pharmacology; 35, 257-274.

Ghosh, M.N., 2011. Fundamentals of Experimental Pharmacology. 3^rd edition, Hliton& Company, Kolkata.

Goyal, R.K., 2007. Practicals in Pharmacology. 8^th edition, B.S. Shah Prakashan, Ahmedabad.

Kulkarni, S.K., 2007. Practical pharmacology and clinical pharmacy. 1^st edition, Vallabh publications, Delhi.

Commonly Used Experimental Animals In Pharmacology Laboratory

Laboratory animals are those animals which can be bred and maintained in the laboratory under suitable conditions by the taking permission of regulatory authorities. Guinea pig has become synonymous to the experimental animals, but many other species are equally useful in the study of drugs. Preclinical studies of experimental pharmacology involve laboratory animals using wide‐ranging doses of the study drug to obtain preliminary efficacy, toxicity and pharmacokinetic information. Such tests assist pharmaceutical companies to decide whether a drug candidate has scientific merit for further development as an investigational new drug. Preclinical study involves the use of laboratory animals. Experimental animals can be classified as:

1.     Rodents- Rat, Mouse, Guinea pig, Hamster etc.

2.     Non rodents- Rabbit, Dog, Cat, Pig, Monkey etc.

3.     Miscellaneous- Frog, Pigeon, chicken etc.

The commonly used laboratory animals in pharmacology laboratory are Frog, Rat, Mice, Guinea pig, Hamster and Rabbit. Cats and dogs are used to study the blood pressure experiments. Monkey, pig and some other animals were also used earlier. But now their use has been restricted. However, Beagle dogs are the only strain approved by USFDA for testing of new drugs.

RAT (Rattus norvegius): 

RAT

Rat is a warm blooded rodent which is the most commonly used animal for biomedical research. Rat does not vomit due to lack of vomiting center and tightly presence of lower esophageal sphincter. It has no tonsils and gall bladder in its body. It has diffuse pancreases, thus difficult to perform pancreatectomy to induce type 1 diabetes mellitus. Fundus and pyloric parts of stomach have clear lining between them and gastric acid secretion is continuous. Rat is a coprophagy animal that eats their own stool. It shows resistance to the effect of Cardiac glycosides. Oestrus cycle appeared at puberty at the age of two to three months and cycle lasts for about four to five days. The cycle can be divided into four stages as follows:
Estrus: It lasts for 9-15 h. The vaginal smear shows cornified epithelial cells. It is characterized by sexual receptivity when the female will allow copulation. It ends with ovulation. It can be induced experimentally by administration of diethylstilbestrol (100 mg/kg).

      Met-estrus: Its about 20 h and occurs shortly after ovulation. The vaginal smear consists of many leucocytes with few cornified epithelial cells.

         Di-estrus: It is the longest phase of about 60-70 h. The vaginal smear shows leucocytes only.

       Proestrus: It lasts for about 12 h. The vaginal smear shows nucleated epithelial cells either singe or in groups.

Wistar rats and Albino Sprague-Dawley rats have been widely used strains throughout the world. Lewis and porton strains are also used for experimental purposes.

MOUSE (Mus musculus):

MOUSE

Mice are worm blooded rodent which are the smallest laboratory animals. In biomedical research mice are preferred over other species because they can be bred uniformly, cheap and easy to handle, require small place for housing and very sensitive to small doses of a drug substances. Due to the large similarity in mice and human genome, it provides good model for research on wide variety of human diseases. Swiss albino mouse is the most widely used strain for pharmacological experiments. Laca and Balb-C strains are also used for experimental purposes.

GUINEA PIG (Cavia porcellus):

GUINEA PIG

Guinea pigs name itself has become synonymous to an experimental animal. Guinea pig is a warm blooded rodent and very docile laboratory animal. It is an herbivorous animal and daily requires vitamin C in diet like human. Guinea pig is highly sensitive to histamine. They are very susceptible to tuberculosis and anaphylactic shock. Penicillin is more toxic to Guinea pig than to the mouse. Guinea pig serum contains an enzyme asparaginase, which has antileukemic action.

HAMSTER (Mesocricetus auratus):

HAMSTER

Hamsters are a brown to gold colour animal, have chunky body with short legs, a diminutive fluffy tail and a large amount of loose skin covered with dense short soft fur. There are four toes on the front foot and five on the back. They have prominent cheek pouches extending up to the shoulder region. The ears are quite prominent, rounded, usually pigmented and scarcely covered with hair. The most common strain used in pharmacological studies is the Syrian or Golden hamster, Chinese hamster, European hamster and American hamster.

RABBIT (Oryctolagus cuniculus):

RABBIT

Rabbit is a worm blooded mammalian and a docile animal used for various studies. Some strains of rabbit are resistant to the effect of atropine because they have higher concentrations of Atropinesterase enzymes in their blood. In rabbits coitus itself induces secretion of leutenising hormone (LH) in females, which leads to ovulation. Progesterone is known to block such ovulation. New Zealand white rabbits are the most common strain used widely in pharmacological studies. Other strains which are also used in studies are the Dutch, the Flemish Giant, Himalayan black and some of the domestic strains.

FROG (Rana tigrina):

FROG

Frog is a cold blooded amphibian which is used very commonly in the biomedical research. Frogs and toads have no taxonomic differences but can be differentiated by their appearances (frog is wet and toad is dry). It has three chambers in its heart, two auricles and one ventricle. The oxygen and moisture are absorbed through their highly permeable skin. Some hybridized frogs are used in biomedical research are Rana esculenta, Rana pipiens, Rana temporaria, Rana lessonae, Rana ridibunda etc.

Table 1: Uses of different laboratory animals in experimental pharmacology.

ANIMALS	USES IN EXPERIMENTAL PHARMACOLOGY
Frog	a)     Commonly used for bio-assays and in pregnancy assays. b)    Different drugs acting on central nervous system, neuro-muscular junction and heart can be studied using frog and its isolated tissues. c)     Whole frog is also used in screening of central drugs like local anesthetics.
Rat	a)     The rat and its isolated tissues are used in the screening of drugs acting on central nervous system, cardiovascular system, estrus cycle, mating behavior, gastric ulcers and acid secretions in stomach. b)    Rats can be trained properly for various types of work performances in response to a reward or punishment, hence suitable for testing of psycho-pharmacological agents. c)  It is also useful in the hormonal assay, immunity, transplantation, immuno-suppression, carcinogenicity study and different toxicity studies. d)   The isolated tissues like ileum, trachea, heart, anacoccygius muscle, stomach strip, vas deferens, uterus and phrenic nerve-diaphragm are used in experimental pharmacology.
Mouse	a)     Mice are the widely used animal in different toxicity studies. b)    Mice are also used in cancer and genetics research, immunity, transplantation related experiments. c)     Screening of analgesics, central nervous system and chemotherapeutics are carried out in this species. d)    Isolated tissues of mice are rarely used (except vas deferens and ileum) in the experimental pharmacology because they are very small and delicate. e)     They were earlier used in the bioassay of insulin.
Guinea pig	a)     It is mainly used in the bio-assay of Histamine. b)    They are used in the study of drugs like broncho-dilators, local anesthetics, spasmolytic and digitalis. c)     The drugs affecting the hearing activity are also screened using Guinea pigs. d)    They are widely used in immunology. e)     The drugs inducing anaphylaxis and hypersensitivity are screened using Guinea pigs. f)     It is also useful in screening of drugs for the treatment of tuberculosis, cholera and amoebiasis. g)    The isolated tissues like ileum, tracheal chain, vas deference and heart are used in experimental pharmacology.
Hamster	a)     The presence of cheek pouch in hamster makes it useful in immunological research. b)    Syrian hamsters are used in the research field of virology, cancer, genetic, toxicology and reproduction. c)     Chinese hamsters are commonly used in diabetes due to deficiency of beta cells, or presence of defective beta cells in the pancreas. d)    Chinese hamsters have a low chromosome number (22) compared to other laboratory animals due to which they are used for cytological investigations, genetics and tissue culture. e)     Hamsters are also used in Onco virus, influenza virus, respiratory syncytial virus studies and vaccine production. f)     Cheek pouch doesn’t have lymphatic drainage and hence they can be used as a site for tissue transplant such as tumors and grafts. g)    The stomach pouch strips of Syrian hamster have been used for the essay of prostaglandins.
Rabbit	a)     Rabbits are mainly used in the pyrogen testing, in bioassays of insulin and sex hormones. b)    They have been used in the screening of drugs for diabetes, diphtheria, tuberculosis, cancer and heart diseases. c)     They are commonly used in genetics, nutrition, toxicology, immunology, physiology and reproduction. d)    They are also useful in the study of teratogenicity of the drugs. e)     Isolated tissues like duodenum, ileum and heart are used in experimental pharmacology. f)     Bi-oassay of Adrenaline can be performed using rabbit ileum. g)    It is an ideal animal for pharmacokinetic studies. h)    Apart from the drugs, effects of creams, cosmetics, special diets and food additives have also been tested in rabbits.
Dog	a)     The dog is used as a model for cardio vascular research, diabetes mellitus, ulcerative colitis, open heart surgery, organ transplantation, central nervous system, pharmacology and toxicology studies etc. b)    Anaesthetized dogs are commonly used in the study of drugs acting on blood pressure and vascular system.
Cat	a)     Cat has distinct nictiating membrane (third eyelid), which is commonly used in screening of ganglionic blocking drugs. b)    Cats are mainly used in behavioral studies, cardio vascular studies, nerve impulse transmission etc. c)     It is also used in neuropharmacology, toxicology, oncology and chromosomal abnormalities studies. d)    It is also employed for the study of drugs effecting on blood pressure.
Monkey	a)     The monkey is used in the fields of virology, parasitology, immunology and immunosuppression. b)    It is also employed for the study of nutrition, reproduction etc. c)     Adult rhesus monkeys are used for N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered model of parkinson’s disease.
Pig	a)     They are used in pharmacological and toxicological research. b)    They are used in important research areas like several isolated organ models, investigation of skin permeation and for digestive systems.
Pigeon	a)     They are used in screening of antiemetic activity, cardiovascular diseases, CNS disorders and intravenous anesthetics. b)    Bioassay of prolactin through the pigeon crop method is one of the important methods.
Chicken	a)     They are used in many areas of biomedical research such as breeding and genetics, growth, embryology, incubation, fertility, artificial insemination, toxicology, physiology, biochemistry, endocrinology and neurobiology. b)    Chick comb method, heart rate, EEG, memory, angiogenesis, blood pressure measurement and vasodilating activities like different models can be developed in chicken.

Table 2: Biological and physiological data of commonly used laboratory animals.

Parameter	Rat	Mouse	Guineapig	Hamster	Rabbit
Adult weight (g)	150-300	20-40	600-800	80-150	1000-3500
Life span (years)	2-3	1.5-2.5	3-5	2-3	4-7
Suitable age for experiment (Months)	1.5	0.75	3	1	6
Gestation period (Days)	21-23	19-21	68-72	16	31-32
Daily food intake (g/ 100 g body weight)	5-10	15-20	6-8	7-10	5-7
Daily water intake (ml/100 g body weight)	10	20	10	10	10
Rectal temperature (ºC)	36-46	38-39	37.2-40	37-38	38.5-40
Heart Rates (beats/ min)	300-500	330-780	230-400	300-500	130-300
Systolic blood pressure (mm Hg)	116	113	77	108	110
Diastolic blood pressure (mm Hg)	90	81	47	77	80
Blood volume (ml/ Kg)	6-7	7-9	6-12	6-9	4-8
Respiratory rate (min)	65-180	84-230	70-104	33-127	38-60

BIBLIOGRAPHY

1.     Ghosh, M.N., 2011. Fundamentals of Experimental Pharmacology. 3^rd edition, Hliton& Company, Kolkata.

2.          Goyal, R.K., 2007. Practicals in Pharmacology. 8^th edition, B.S. Shah Prakashan, Ahmedabad.

3.          Kulkarni, S.K., 2007. Practical pharmacology and clinical pharmacy. 1^st edition, Vallabh publications, Delhi.

4.      Medhi, B., Prakash, A., 2010. Practical Manual of Experimental and Clinical Pharmacology. 1^st edition, Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India.

5.          Rahman, S.Z., Salman, M.T., 2009. A guide to the alternatives to animal experimentation. 1^st edition, Muslim Educational Press, Aligarh.

6.        Vogel, H.G., 2002. Drug Discovery and Evaluation Pharmacological Assays. 2^nd edition, Springer-Verlag, Berlin, Heidelberg, New York.