CPCSEA Guidelines ,GLP & Bioassay.pdf

The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) was established under Section 15(1) of the Prevention of Cruelty to Animals Act 1960.
GLP is a quality system concerned with the organizational process and conditions under which non-clinical health and environmental safety studies are planned, performed, monitored, recorded, archived and reported. GLP principles include. Organization and Personnel. Management-Responsibilities.
A bioassay is an analytical method to determine the concentration or potency of a substance by its effect on living animals or plants, or on living cells or tissues. A bioassay can be either quantal or quantitative, direct or indirect.

1. CPCSEA , GLP & BIOASSAY
Present By-
Mr. Dnyaneshwar Balu Gutale
M. Pharmacy (Pharmacology)
Vidyabharti College of Pharmacy Amravati

2. Content
•CPCSE Introduction
•CPCSEA Guidelines
•GLP
•GLP Principles
•Bioassay
•Principle of Bioassay
•Types Of Bioassay

3. CPCSEA
•Committee for the Purpose of Control and Supervision of Experiments
on Animals (CPCSEA)
•Statutory body formed by the Act of the Indian Parliament under the
Prevention of Cruelty to Animals Act, 1960
•Formed in 1964
•Revived in 1998, under the committed chairpersonship of Meneka
Gandhi
•Head quarters at Chennai
•Includes various guidelines and sub committees

4. •National level expert committee to scrutinize and approve the
research projects conducted using animals.
•Introduced a national document “Good Laboratory Practice”
•2004-CPCSEA officially accepts the concept of the 4
th
R
•“investigators have a moral responsibility to take care of laboratory
animals after use”
This committee is composed of-
•Members of the scientific community
•Regulatory authorities
•Animal activists.
•The committee has 10 experts and 3 official membersresponsibility

5. Goals:
•To promote the humane care of animals used in research.
•To provide specifications that will enhance animals well being and
quality of research.
Objective:
•Effective functioning of Institutional animal ethical committee

6. Main Activities
•Registration of establishments
1.for experiments on animals
2.for Breeding of animals
•Approval of Animal House Facilities
•Permission of Committee for Conducting Experiments

7. CPCSEA Guidelines
Veterinary care:
•Provided by a veterinarian
•Daily observation of animals adopted
Quarantine, Stabilization and Separation:
• Quarantine period -
1.Small lab animals: one wk to one mth
2.Large lab animals: up to 6 weeks
• Physiologic, psychological and nutritional stabilization required
• Physical separation of animals by species to prevent anxiety and behavioral changes

8. Animal Procurement & Transportation:
•Procurement of animals done from established commercial animal vendors.
•During transport, stress avoided by :-
✓Use of transport containers (cages or crates) of appropriate size
✓Permissible number of animals accommodated in each container
Experimental area:
•Experiments should be carried out in a separate area away from the place where
they are housed.
•Separate functional areas for-
✓surgical support
✓treatment of animals
✓post operative and intensive care

9. Physical Facilities
Building Materials Durable, moisture-proof, fire resistant and pest
resistant
Corridor(s) Wide enough to facilitate the movement of
personnel and equipment
Animal room doors Should fit properly, should not be rust
Exterior windows Windows are not recommended
Floors Smooth, moisture proof, nonabsorbent, skid
proof floors
Drains Proper drainage, Floors should be sloped
Storage areas Designed for feed, bedding, cages and materials
not in use.

10. Environment
Temperature Between 18-29°C
Humidity control Range of 30-70%
Ventilation Designed with 12-15 air cycles per hour.
Power and lighting
System should be safe Fluorescent lights are
efficient Emergency backup needed
Noise control
Noise free environment Concrete walls are
more effective

11. Animal Husbandry
Caging or Housing System
The housing system should provide-
•Adequate space, freedom of movement,normal postural adjustments
•Comfortable environment
•Easy access to food and water
•Adequate ventilation
Food & Water
•Food should be palatable, non-contaminated, and nutritionally adequate
•Feed should contain –Moisture ,Ocrude fibre, crude protein and crude fat,
essential bits, minerals, carbohydrates
•Feed should not contain -Insecticides, hormones, antibiotics, fumigants or
potential toxicants,Heavy metals.
•Should have continuous access to fresh, potable uncontaminated drinking water

12. Bedding
•Should be absorbent, free from toxic chemicals
•Should be removed and replaced periodically with fresh materials
•Ideal to change the bedding twice a week.
Sanitation and cleanliness
•Animal rooms, corridors, storage spaces, and other areas should be cleaned
with appropriate detergents and disinfectants
•Wastes should be removed regularly and frequently (waste disposal –
incineration)
•Cages should be sanitized before animals are placed in them

13. •Proper maintence of records must
•Should maintain the SOP’s
•Proper selection of animal facility staff required
•Anaesthesia : Should be complete and without any pain
•Euthanasia : Done when animal required to be sacrificed or
termination of an experiment or otherwise for ethical reasons

14. GLP
Good Laboratory Practice :-
Good Laboratory Practice is a quality system concerned with the
organizational process and the conditions under which a study is
planned, performed, monitored, recorded, archived and reported.
GLP applies to nonclinical studies conducted for the assessment of the
safety or efficacy of chemicals (including pharmaceuticals).
GLP helps assure regulatory authorities that the data submitted are a
true.

15. HISTORY
•The formal regulatory concept of “Good Laboratory Practice” (GLP)
originated in the USA in 1970’s.
•The FDA’s publication of Proposed Regulations on GLP in 1976, with
establishment of the Final Rule in June 1979 (21 CFR 58).
•In 1981 an organization named OECD produced GLP principles that
are international standard.

16. OBJECTIVES OF GLP
•GLP makes sure that the data submitted are a true reflection of the results that
are obtained during the study.
•GLP also makes sure that data is traceable.
•Promotes international acceptance of tests.
MISSION OF GLP
•Test systems
•Archiving of records.
•Apparatus, material and reagent facilities.
•Quality assurance programs
•Performance of the study.
•Reporting of study results.
•Standard operating procedures (SOP)

17. GLP Principles
1. Test facility organization and personnel
2. Quality Assurance (QA) program
3. Facilities
4. Apparatus materials and reagents
5. Test systems
6. Test and reference items
7. Standard Operating Procedures (SOP’s)
8. Performance of the study
9. Reporting of study results
10. Storage and retention of records and materials

18. 1.Test Facility Organization and Personnel
Study Personnel Responsibilities
•Should have the Knowledge of the GLP principles.
•Access to the study plan and appropriate SOP’s.
•Comply with the instructions of the SOP’s.
•Record raw data.
•Study personnel are responsible for the quality of their data.
•Exercise health precautions to minimize risk
•Ensure the integrity of the study.

19. 2.Quality Assurance Program
Responsibilities of the QA Personnel
•Access to the updated study plans and SOP’s.
•Documented verification of the compliance of study plan to the GLP
principles.
•Inspections to determine compliance of the study with GLP principles.
•Three types of inspection.
-Study-based inspections.
-Facility-based inspections.
-Process-based inspections.
•Inspection of the final reports for accurate and full description.
•Report the inspection results to the management.
•Statements.

20. 3. Facilities
•Suitable size, construction and location.
•Adequate degree of separation of the different activities.
•Isolation of test systems and individual projects to protect from biological
hazards.
•Suitable rooms for the diagnosis, treatment and control of diseases.
•Storage rooms.
4. Apparatus, Materials and Reagents
•Apparatus of appropriate design and adequate capacity.
•Documented Inspection, cleaning, maintenance and calibration of
apparatus.
•Apparatus and materials not to interfere with the test systems.
•Chemicals, reagent and solutions should be labeled to indicate identity,
expiry and specific storage instructions.

21. 5. Test Systems
•Physical and chemical test systems.
•Biological test systems.
•Records of source, date of arrival, and arrival conditions of test systems.
•Proper identification of test systems in their container or when removed.
•Cleaning and sanitization of containers.
•Pest control agents to be documented.
6. Test and Reference Items
•Receipt, handling, sampling and storage
•Characterization.
•Known stability of test and reference items.
•Stability of the test item in its vehicle (container).
•Experiments to determine stability in tank mixers used in the field studies.
•Samples for analytical purposes for each batch.

22. 7.Standard Operating Procedures (SOP)
•Written procedures for a laboratories program.
•They define how to carry out protocol-specified activities.
•Most often written in a chronological listing of action steps.
•They are written to explain how the procedures are suppose to work.
•Routine inspection, cleaning, maintenance, testing and calibration.
•Actions to be taken in response to equipment failure.
•Keeping records, reporting, storage, mixing, and retrieval of data.
•Definition of raw data.
•Analytical methods.
8. Performance of the Study
•Prepare the Study plan.
•Content of the study plan –Identification of thefacility, Records, Dates, Reference to test
methods, Information concerning the sponsor and facility.
•Conduct of the study.

23. 9. Reporting of Study Results
•Information on sponsor and test facility.
•Experimental starting and completion dates.
•A Quality Assurance Program Statement.
•Description of materials and test methods.
•Results.
•Storage (samples, reference items, raw data, final reports) etc.
10. Storage and Retention of Records and Materials
•The study plan, raw data, samples.
•Inspection data and master schedules.
•SOPs.
•Maintenance and calibration data.
•If any study material is disposed of before expiry the reason to be justified and documented.
•Index of materials retained.

24. What Good Laboratory Must Contain.?
•Area should be free from smoke, smell, dust etc.
•Ensure good ventilation, proper illumination and prefer natural light.
Air conditioned the lab with humidity control.
•Enough space for measuring and testing instrument.
•Proper arrangement of testing.
•Take care of all safety points including proper earthing as well as fire
safety.
•Avoid uncleanable spots in floors, walls, ceiling.
•Establish proper areas for storage of incoming samples as well as test-
completed samples.
•Also provide sample collection place as well as packing and disposal
of tested samples.

25. Bioassay
•Bioassay is defined as the estimation of the potency of an active principle
in a unit quantity of preparation.
OR
•Detection and measurement of the concentration of the substance in a
preparation using biological methods.
PRINCIPLE OF BIOASSAY
The basic principle of bioassay is to compare the test substance with the
International Standard preparation of the same and to find out how much
test substance is required to produce the same biological effect, as produced
by the standard.

26. TYPES OF BIOASSAY
1.Quantal
2.Graded
1.QUANTAL ASSAY
Quantal response:
•The response is in the form of ‘all or none’, i.e. either no response or
maximum response.
•Drugs producing quantal effect can be bioassayed by End-point
method.Quantal

27. •The threshold dose producing a predetermined effect is measured
•Comparison between the results of standard and the test
•E.g: Bioassay of digitalis in cats, Insulin induced hypoglycemic convulsions in rat
2. GRADED ASSAY
Graded response:
Response is proportional to the dose and response may lie between no response and the maximum
response.
Types:
•Bracketing /direct matching
•Interpolation
•Multiple Point Assay-Three point assay, Four point assay, Six point assay
•Cumulative dose response

28. Bracketing or Direct Matching
•A constant dose of the standard is bracketed by varying dose of test sample
•until an exact matching between the response of std & that of the sample
is achieved
•Strength of unknowm/test drug can be found by simple interpolation of
bracketed response.
Interpolation assay
•A log dose-response curve is plotted with the standard on a simple graph
paper or Semi-log paper
•The concentration of the test is then read from the graph

29. Multiple point assay
•Responses are repeated several times and the mean of each is taken
•Chances of error are minimized
3 point method -2 doses of std+1 dose of test
4 point method –2 doses of std+2 doses of test
6 point method –3 doses of std+3 doses of test
•Latin square method of randomization to avoid any bias

30. 3 Point Assay-
Calculation
•Mean responses of these 3 sets plotted
•Log potency ratio (M) = (T-S1: S2-S1)x log d
•where, d-dose ratio = s2/s1
•Strength of unknown = s1/tx antilog of M

31. 4 Point Assay-
Calculation
•Mean responses of 4 sets plotted Log potency ratio (M)
[(T2-52)+(T1-51) /(S2-S1)+(T2-T1)] ×Log d
where, d-dose ratio = $2/s1
•Strength of unknown = s1/t1 x antilog of M

32. Six point assay
•3+3 dose assay
•3 conc each of std & test drug are used
•6 sets of experiments using 6 doses in each set
•More time consuming, lesser in use Reliability is excellent