Timeline of the rDNA guidelines

i. The Government of India came out with the “Rules for the Manufacture/Use/Import/Export
and Storage of Hazardous Microorganisms, Genetically Engineered Organisms or Cells” on 5th
December, 1989 under the provisions of Environment (Protection) Act, 1986 through the
Ministry of Environment & Forests (MoEF).
ii. These rules are commonly referred as ‘Rules 1989’.
iii. The two main agencies identified for implementation of the Rules 1989 are the Ministry of
Environment & Forests and the Department of Biotechnology (DBT), Ministry of Science and
Technology, Government of India. The Rules 1989 have also defined Competent Authorities
and the composition of such Authorities for handling of various biosafety aspects.
iv. For implementation of the Rules 1989, DBT has brought out Guidelines from time to time.
In 1990, the Guidelines under the title “Recombinant DNA Safety Guidelines” were
published.
 Rules for the manufacture, use/import/export and
storage of hazardous micro organisms/ genetically engineered
organisms or cells

In India, the regulation of all activities related to GMOs and products derived from GMOs is
governed by “Rules for the Manufacture/Use/Import/Export and Storage of Hazardous
Microorganisms, Genetically Engineered Organisms or Cells, 1989” under the provisions of the
Environment (Protection) Act, 1986 through the Ministry of Environment and Forests (MoEF). This
act came into provision on 5th December 1989.
 1. Scope of the guidelines
i. The guidelines deals with genetic transformation of green plants, rDNA technology in
vaccine development and on large scale production and deliberate/ accidental release
of organisms, plants, animals and products derived by rDNA technology into the
environment
ii. While preparing the revised guidelines the Committee and its sub-groups have met 4
times and have taken note of the guidelines currently in use in other countries. (the
guidelines have been modified further in 1994)
iii. The evolution of the guidelines and updation have gone through the process of
consultation with experts, academies, agencies and industry and the concerned
Ministries with a view to gain general acceptance and broad consensus
iv. The guidelines are in respect of safety measures for the research activities, large scale
use and also the environmental impact during field applications of genetically altered
material products.
v. Exceptions to the guidelines: Isuues relating to genetic engineering of human embryo,
use of embryos and fetuses in research and human germ line, and gene therapy areas.
 2. Competent Authorities

6 Statutory bodies have been designated in order to implement the 1989 guidelines. These are the
same bodies that implement the modified guidelines.
They aare:
i. Recombinant DNA Advisory Committee (RDAC)
ii. Institutional Biosafety Committee (IBSC)
iii. Review Committee on Genetic Manipulation (RCGM)
iv. Genetic Engineering Approval Committee (GEAC)
v. State Biotechnology Coordination Committee (SBCC)
vi. District Level Committee (DLC)

While the RDAC is of advisory in function, the IBSC, RCGM, and GEAC are of regulatory function, SBCC
and DLC are for monitoring purposes.
To summarize, under Rules, 1989, IBSC, RCGM and GEAC are involved in approval process of
LMOs/GMOs and SBCC and DLC have monitoring functions. The procedures involved in the approval
of GMOs in India are summarized below:
3. Definitions

(i) “Biotechnology” means the application of scientific and engineering principles to the processing
of materials by biological agents to produce goods and services;
(ii) “Cell hybridisation” means the formation of live cells with new combinations of genetic
material through the fusion of two or more cells by means of methods which do not occur
naturally;
(iii) “Gene Technology” means the application of the gene technique called genetic engineering,
include selfcloning and deletion as well as cell hybridisation;
(iv) “Genetic engineering” means the technique by which heritable material, which does not usually
occur or will not occur naturally in the organism or cell concerned, generated outside the
organism or the cell is inserted into said cell or organism. It shall also mean the formation of new
combinations of genetic material by incorporation of a cell into a host cell, where they occur
naturally (self cloning) as well as modification of an organism or in a cell by deletion and
removal of parts of the heritable material;
(v) “microorganisms” shall include all the bacteria, viruses, fungi, mycoplasma, cell lines, algae,
protozoans and nematodes indicated in the schedule and those that have not been presently
know to exist in the country or not have been discovered so far.
4.Classification of microorganisms or genetically
engineered product

(i) For the purpose of these rules, microorganisms or genetically engineered

organisms, products or cells shall be dealt with under two major heads;
animal pathogens and plant pests and these shall be classified in the manner
specified in the Schedule.
(ii) If any of the microorganism, genetically engineered organism or cell falls
within the limits of more than one risk class as specified in the Schedule, it
shall be deemed to belong exclusively to the last in number of such
classes.

5. Microorganisms laid down in the schedule are divided into the following
subcategories
i. Bacterial agents:
ii. Fungal agents:
iii. Parasitic agents
iv. Viral, rickettsial and chlamydial agents:
v. Special category
5. Production
Production in which genetically engineered organisms or cells or micro-
organism are generated or used shall not be commenced except with the
consent of genetic engineering approval committee with respect of discharge
of genetically engineered organisms or cells into the environment. This shall
also apply to production taking place in connection with development, testing
and experiments where such production, etc, is not subject to rule 7.

6. Deliberate or unintentional release

(1) Deliberate or unintentional release of genetically engineered
organisms/hazardous microorganisms or cells, including deliberate release
for the purpose of experiment shall not be allowed.
Note: deliberate release shall mean any intentional transfer of genetically
engineered organisms/hazardous microorganisms or cells to the
environment or nature, irrespective of the way in which it is done:
(2) the genetic engineering approval committee may in special cases give
approval of deliberate release.

7. Permission and approval for certain substances

Substances and products, which contain genetically engineered organisms
or cells or microorganisms shall not be produced, sold, imported or used
except with the approval of genetic engineering approval committee
8. Permission and approval for food stuffs
Food stuffs, ingredients in food stuffs and additives including processing
aids containing or consisting of genetically engineered organisms or cells,
shall not be produced, sold, imported or used except with the approval of
the genetic engineering approval committee.

9. Supervision

(1) The Genetic Engineering Approval Committee may supervise the

implementation of the terms and conditions laid down in connection
with the approvals accorded by it.
(2) The Genetic Engineering Approval Committee may carryout this
supervision through the State Biotechnology Coordination Commit-tee
or the State Pollution Control Boards/District Level Commit-tee or
through any person authorised in this behalf.
(3)All approvals of the Genetic Engineering Approval Committee shall be for
a specified period not exceeding four years
10. Penalties

(1) If an order is not complied with, the district level committee or state
biotechnology co-ordination committee may take measures at the
expenses of the person who is responsible.
(2) In cases where immediate interventions is required in order to prevent
any damage to the environment, nature or health, the district level
committee or state biotechnology coordination committee may take the
necessary steps without issuing any orders or notice. The expenses
incurred for this purpose will be repay-able by the person responsible for
such damage.
(3) The state biotechnology co-ordination committee /district level
committee may take samples for a more detailed examination of
organisms and cells.
(4) The state biotechnology co-ordination committee/district level
committee shall be competent to ask for assistance from any other
government authority to carry out its instructions.
11. Responsibility to notify interruptions or accidents
(1)Any person who under rule 7-11 is responsible for conditions or arrangements shall
immediately notify the district level commit-tee state biotechnology co-ordination committee
and the state medical officer of any interruption of operations or accidents that may lead to
discharges of genetically engineered organisms or cells which may be harmful to the
environment, nature or health or involve any danger thereto.
(2)Any notice given under sub-rule (1) above shall not lessen the duty of the person who is
responsible to try effectively to minimise or prevent the effects of interruptions of operations of
accidents.

12. Preparation of off-site emergency plan by the DLC

It shall be the duty of the DLC to prepare an off-site emergency plan detailing how emergencies
relating to a possible major accident at a site will be dealt with and in preparing the plan, the
DLC shall consult the occupier and such other person as it may deem necessary

13. Inspections and informations regarding finance

The state biotechnology co-ordination committee or the genetic engi-neering approval
committee/the DLC or any person with special knowledge duly authorised by the state
biotechnology co-ordina-tion committee or the genetic engineering approval committee or the
DLC where it is deemed necessary, at any time on due produc-tion if identity be admitted to
public as well as to private premises and localities for the purpose of carrying out supervision.
1. Terms and definitions associated with
the guidelines
A. Recombinant DNA :
i. In vitro introduction of different segments of DNA that are
capable of replication in a host cell either autonomously or as an
integral part of host's genome and maintenance of their
continued propagation.
ii. This will include all types of cell fusion, microinjection of DNA or
RNA or parts or all of chromosomes, genetic engineering including
self cloning and deletion as well as cell hybridization,
transformation and other types of virus or pathogen introduction
into unnatural hosts.

B. Containment:
The safe methods used for managing infectious agents in the
laboratory environment where they are being handled or
maintained in order to reduce exposure of laboratory workers,
other persons, and the outside environment to potentially
Types of Containment
 Biological containment (BC): Any combination of vector and host
which is to provide biological containment must be chosen or
constructed to limit the infectivity of vector to specific hosts and
control the host-vector survival in the environment

 Physical Containment (PC): The objective of physical containment is

to confine recombinant organisms thereby preventing the exposure of
the researcher and the environment to the harmful agents. Physical
containment is achieved through the use of
i) Laboratory Practice,
ii) Containment Equipment, and
iii) Special Laboratory Design
Elements of Containment:
Laboratory practice and technique:
 Strict adherence to standard microbiological practices and techniques
 Awareness of potential hazards
 Providing/arranging for appropriate training of personnel
 Selection of safety practices in addition to standard laboratory
practices if required
 Developing and adopting biosafety or operations manual which
identifies the hazards
Safety equipment (primary barriers):
 Safety equipment includes biological safety cabinets and a variety of
enclosed containers (e.g. safety centrifuge cup).
 The biological safety cabinet (BSC) is the principal device used to
provide containment of infectious aerosols generated by many
microbiological procedures.
 Three types of BSCs (Class I, II, III) are used in microbiological
laboratories. Safety equipment also includes items for personal
protection such as gloves, coats, gowns, shoe covers, boots,
respirators, face shields and safety glasses, etc.
Facility Design (Secondary barriers):
 The design of the facility is important in providing a barrier to
protect persons working in the facility
 But outside of the laboratory and those in the community from
infectious agents which may be accidentally released from the
laboratory.
 There are three types of facility designs:
i) The Basic Laboratory (for Risk Group I and II),
ii) The Containment Laboratory (for Risk Group III)
iii) The Maximum Containment Laboratory (for Risk Group IV)
Biosafety levels and the Biosafety Cabinet
 It consists of a combination of laboratory practices and techniques,
safety equipment and laboratory facilities appropriate for the
operations performed and the hazard posed by the infectious
agents.
 The guidelines for Microbiological and Biomedical Laboratories
suggest four Biosafety levels in incremental order depending on
the nature of work.

A typical Class I, Class II and Class III Biosafety Cabinets (from left)
Categorization of microorganisms
based on pathogenicity
Hazard Organisms that are most unlikely to cause human disease
Group 1
Hazard Organisms capable of causing human disease and which
Group 2 may be a hazard to laboratory workers, but are unlikely to
spread to the community. Laboratory exposure rarely
produces infection and effective prophylaxis or effective
treatment is usually available
Hazard Organisms that may cause severe human disease and
Group 3 present a serious hazard to laboratory workers. They may
present a risk of spread to the community, but there is
usually effective prophylaxis or treatment available
Hazard Organisms that cause severe human disease and are a
Group 4 serious hazard to laboratory workers. They may present a
high risk of spread to the community, and there is usually
no effective prophylaxis or treatment
Summary of recommended Biosafety Levels
for Infectious Agents
Biosafety Practice and Safety Facilities
Level Techniques
1. Standard Non primary containment Basic
microbiological provided by adherence to
practices standard laboratory
practices
2. Level 1 practices plus Partial containment Basic.
laboratory coats; equipment (i.e. Class I or
decontamination of all II Biological Safety
infectious wastes Cabinets) used to
limited access; conduct mechanical and
protective gloves and manipulative procedures
biohazard warning that have aerosol
signs as indicated potential that may
increase the risk of
exposure to personnel
Biosafety Practice and Safety Facilities
Level Techniques
3. Level 2 practice plus Partial containment Containment
special laboratory equipment used for all
clothing, controlled manipulations of
access infectious material
4. Level 3 practices plus Maximum containment Maximum
entrance through equipment (i.e. class III containment
change room where biological safety cabinet
street clothing is or partial containment
removed and equipment in combination
laboratory clothing is with full body air supplied,
put on shower on exit, positive pressure
all wastes are personnel suit used for all
decontaminated on procedures and activities
exit from the facility
Bio-safety levels
Biosafety Level 1:
 These practices, safety equipment and facilities are appropriate for
undergraduate and secondary educational training and teaching
laboratories and for other facilities in which work is done with defined
and characterized strains of viable microorganisms not known to cause
disease in healthy adult human.
 No special accommodation or equipment is required but the laboratory
personnel are required to have specific training and to be supervised
by a scientist with general training in microbiology or a related
science.
Biosafety Level 2:
 These practices, safety equipment and facilities are applicable in
clinical, diagnostic, teaching and other facilities in which work is done
with the broad spectrum of indigenous moderate-risk agents present in
the community and associated with human disease of varying severity.
 Laboratory workers are required to have specific training in handling
pathogenic agents and to be supervised by competent scientists.
 Accommodation and facilities including safety cabinets are prescribed,
Biosafety level 3:
 These practices, safety equipment and facilities are applicable to clinical,
diagnostic, teaching research or production facilities in which work is done with
indigenous or exotic agents where the potential for infection by aerosols is real
and the disease may have serious or lethal consequences.
 Personnel are required to have specific training in work with these agents and
to be supervised by scientists experienced in this kind of microbiology.
 Specially designed laboratories and precautions including the use of safety
cabinets are prescribed and the access is strictly controlled.

Biosafety level 4:
 These practices, safety equipment and facilities are applicable to work with
dangerous and exotic agents which pose a high individual risk of life-
threatening disease.
 Strict training and supervision are required and the work is done in specially
designed laboratories under stringent safety conditions, including the use of
safety cabinets and positive pressure personnel suits .
 Access is strictly limited.
2. Classification of pathogenic
microorganisms
The classification of infective microorganisms are drawn up under 4
risk groups in increasing order of risk based on the following
parameters:
 Pathogenecity of the agent
 Modes of transmission and host range of the agent
 Availability of effective preventive treatments or curative medicines
 Capability to cause diseases to humans/animals/plants
 Epidemic causing strains in India

The scientific considerations for assessment of potential risks in

handling of pathogenic organisms include the following:
 Characterization of donor and recipient organisms
 Characterization of the modified organism
 Expression and properties of the gene product
3. Categories for rDNA research activities

The guidelines stipulate three categories of research activities, These are:

Category I: Which are exempt for the purpose of intimation and approval of
competent authority.
(i) The experiments involving self cloning, using strains and also inter-species
cloning belonging to organism in the same exchanger group
(ii) Organelle DNA including those from chloroplasts and mitochondria.
(iii) Host-vector systems consisting of cells in culture and vectors, either non-
viral or viral containing defective viral genomes (except from cells known to
harbour class III, IV and special category etiologic agents).
Category II: Those requiring prior intimation of competent authority.
(i) Experiments falling under containment levels II, III and IV.
(ii) Experiment wherein DNA or RNA molecules derived from any source except
for eukaryotic viral genome may be transferred to any non-human vertebrate or
any invertebrate organisms and propagated under conditions of physical
containment PC1 and appropriate to organism under study.
(iii) Experiments involving non pathogen DNA vector systems and regeneration
from single cells.
(iv) Large scale use of recombinants made by self cloning in systems belonging to
exempt category (e.g. E.coli, Saccharomyces, and B. subtilis)
 Category III: Those requiring review and approval of competent
authority before commencement
 Toxin gene clonings
 Cloning of genes for vaccine production
 Cloning of mosquito and tick DNA experiments should be
prescribed on a case by case basis since these are natural vectors
for certain endemic viral and parasitic diseases
 Genes coding for antibiotic resistance into pathogenic organisms
which do not naturally possess such resistance.
 Introduction into cultured human cells of recombinant DNA
molecules containing complete genes of potentially oncogenic
viruses or transformed cellular genes.
 Experiments involving engineered microbes with deletions and
certain rearrangements
4. Large scale experiments
 Experiments beyond 20 litres capacity for research as well as industrial purposes
are included in the category of large scale experimentation/operations.

 For such activities it is recommended that one should seek approval of the
competent authority

 In order to seek approval it will be necessary to furnish the relevant details in a

prescribed format on the lines suggested by Genetic Engineering Approval
Committee (GEAC).

 In case of plants, this limits is beyond 20 acres area.

 The guideline gives principles of occupational safety and hygiene for large-scale
practice and containment.

 Physical containment conditions that should be ensured for large-scale

experiments and production have been specified in the guidelines.
Good large scale practice (GLSP)

 To keep work place and environment exposure to any physical,

chemical or biological agent to the lowest practicable level;
 To exercise engineering control measures at source and to
supplement these with appropriate personal protective clothing
and equipment when necessary ;
 To test adequately and maintain control measures and equipment
 To test when necessary for the presence of viable process
organisms outside the primary physical containment
 To provide training of personnel
 To formulate and implement local code of practice for the safety
of personnel.
Safety criteria for GLSP
 The host organism should not be a pathogen, should not contain
adventitious agents, and should have an extended history of safe
use, or have built-in environmental limitations that permit
optimum growth in the bioreactor but limited survival with no
adverse consequences in the environment.
 The vector/insert should be well characterized and free from
known harmful sequences; the DNA should be limited in size as
much as possible to perform the intended function; should not
increase the stability of the recombinant in the environment unless
that is a requirement of the intended function.
 The genetically manipulated organism should not be a pathogen
and should be assessed as being as safe in the bio-reactor as the
host organism, and without adverse consequences in the
environment
5. Release to the environment
 Depending on the types of organisms handled and assessment of potential risks
involved appropriate containment facilities must be provided to ensure safety of
worker and to prevent unwanted release in the environment.
 Biowastes resulting from laboratory experiments, in industrial operations should
be properly treated so that the pathogenicity of genetically engineered organisms
are either destroyed or rendered harmless before disposal in the environment.
 Special facilities should be created for disposal of experimental animals. All
refuse and carcasses must be incinerated.

For planned release the following points should be considered.

i. Geographical location, size and nature of the site of release and physical and
biological proximity to man and other significant biota. In case of plants,
proximity to plants which might be cross pollinated.
ii. Details of target ecosystem and the predicted effects of release on that
ecosystem.
iii. Method and amount of release, rate frequency and duration of application.
iv. Monitoring capabilities and intentions: how many novel organisms be traced,
e.g. to measure effectiveness of application.
v. Onsite worker safety procedures and facilities.
vi. Contingency plans in event of unanticipated effects of novel organisms.
6. Import and shipment

 The import or receipt of etiologic agents and

vectors of human and animal disease or their
carriers is subject to the quarantine regulations.
 Permits authorizing the import or receipt of
regulated materials for research (e.g. toxin genes,
hybridomas, cell cultures, organelle) and specifying
conditions under which the agent or vector is
shipped, handled and used are issued by the Review
Committee on Genetic Manipulation
 Large scale imports for industrial use are regulated
by Genetic Engineering Approval Committee
 Safety testing may be required to ensure that it is
far from risk.
7. Quality control of biologicals produced by
rDNA technology
i. The general regulations normally applicable for biologicals are
applicable to the recombinant DNA products.
ii. The specific relevant aspects to a particular product should be
discussed with the appropriate Government Agency on a case by
case basis.
iii. A new license for the product or drug application would be
required on products made of recombinant DNA technology
iv. A recombinant DNA product demonstrated to be identical to
normally occurring substance would not require toxicological and
pharmacological data if the information is already available
v. The products derived from animals for human consumption such
as meat and milk should be free from any contaminants or
residue effect resultant on the use of feed stuffs containing
additives produced by biotechnological processes.
8. Mechanism of implementation of biosafety
guidelines

 For implementation of the guidelines it is necessary to have an

institutional mechanism to ensure the compliance of requisite
safeguards at various levels.
 The guidelines prescribe specific actions that include establishing
Safety procedures for rDNA research, production and release to the
environment and setting up containment conditions for certain
experiments.
 The guidelines suggest compliance of the safeguards through
voluntary as well as regulatory approach.
 The 6 competent bodies each play a definite role in implementing
these guidelines.
Summary of the guidelines and how are they
enforced
The approvals and prohibitions under Rules 1989 are summarized below:
➢ No person shall import, export, transport, manufacture, process, use
or sell any GMOs, substances or cells except with the approval of the
GEAC.
➢ Use of pathogenic organisms or GMOs or cells for research purpose
shall be allowed under the Notification, 1989 of the EPA, 1986.
➢ Any person operating or using GMOs for scale up or pilot operations
shall have to obtain permission from GEAC.
➢ For purpose of education, experiments on GMOs IBSC can look after, as
per the guidelines of the Government of India.
➢ Deliberate or unintentional release of GMOs not allowed.
➢ Production in which GMOs are generated or used shall not be
commenced except with the approval of GEAC
➢ GEAC supervises the implementation of rules and guidelines.
GEAC carries out supervision through SBCC, DLC or any authorized
person.
➢ If orders are not complied, SBCC/DLC may take suitable measures
at the expenses of the person who is responsible.
➢ In case of immediate interventions to prevent any damage, SBCC
and DLC can take suitable measures and the expenses incurred will
be recovered from the person responsible.
➢ All approvals shall be for a period of 4 years at first instance
renewable for 2 years at a time.
➢ GEAC shall have powers to revoke approvals in case of:
i. Any new information on harmful effects of GMOs.
ii. GMOs cause such damage to the environment as could not be
envisaged when
iii. Non-compliance of any conditions stipulated by GEAC.
WHO guidelines

i. Premises and lab:

1. Appropriate containment: code of practice; lab design and

facilities; health and medical surveillance; specification for gene
technology lab; specification for large scale operation
2. Prevention against entry of pests (air pressure, exhaust air, input
air)
3. Provisions for emergency
4. Provisions for storage and disposal: In process material; starting
material; finished product; infected material/rejected
5. Cleanliness and hygiene
6. Repair facilities
 Equipment:

1. Adequacy of equipment: appropriate design; set up

and maintenance
2. Standard Operating Procedures (SOP): validation of
all equipment; calibration of all instruments;
investigating recording all deviations and expertise
3. Automated equipment: computer controlled
system; back-up file maintenance and hard copy
systems
 Animal facilities:

1.. Receipt of animals, including identification of person

responsible and required documentation; maintenance,
evaluation of health status; housing, feeding, handling;
isolation of sick animals, preventive measures,
treatment and quarantine for newly received animals

2. Pest control system; facilities for waste, carcass;

cleaning, sterilization and maintenance of supplies and
equipment (animal cages, racks)
 Environment:

SOPs to minimize contamination; monitoring frequency; methods for

viable counts in air, water, surface and non viable particulates in air.