1. INTRODUCTION
2. CONSTITUTION
3. COMPOSITION
4. MEETINGS
6. FUNCTIONS
6.2 IBSC members
6.3 DBT Nominee
7.1 Research
7.2 Large scale trials and
production
I.
Information to IBSC/ RCGM for Import/ Exchange of
GMOs and products thereof for research purpose.
III. Information to IBSC/ RCGM to carry out research for
development of transgenic plants.
IV.
Half yearly report of the Institutional Biosafety
Committee to RCGM.
In view of rapid developments in both
research and commercialization of modern biotechnology, there are concerns with
respect to the impact of genetically modified organisms (GMOs) on human health
and environment. Therefore, it is
extremely important that research programmes, field trials and commercial
activities involving GMOs are monitored right from the time of initiation for
due assessment of risks and incorporation of required management measures as
per the regulations in the country.
Rules for the manufacture,
use/import/export and storage of hazardous microorganisms/genetically
engineered organisms or cells, 1989 notified by the Ministry of Environment
& Forests, Government of India under Environmental Protection Act (1986)
require the constitution of an Institutional Biosafety Committee (IBSC) by
every organization engaged in research and production activities related to
GMOs. Functions of IBSCs have been
elaborated in the “Recombinant DNA Safety Guidelines, 1990” and “Revised
guidelines for research in transgenic plants & guidelines for toxicity and
allergenicity evaluation of transgenic seeds, plants and plant parts, 1998”
issued by the Department of Biotechnology.
An IBSC consists of the head of the
institution or nominee as chairman, scientists specializing in the concerned
discipline, an outside expert and a representative of the medical profession as
members. All the IBSCs should have also
a nominee appointed by DBT.
The IBSC is the nodal point for
interaction within the institution for implementation of the biosafety regulatory
framework. With the increasing research activities on GMOs, it was felt that
the functioning of the committees and their feedback to DBT should be
strengthened by defining the role of the committees and their members in
greater depth and detail by a consultative process. With this in view, DBT in association with Biotech Consortium
India Limited (BCIL) organized a series of national consultations at six
locations namely Bangalore, Chennai, Hyderabad, New Delhi, Mumbai and Jalna
involving the present members of various IBSCs and the DBT nominees. A detailed background document was prepared
and circulated in the meetings. The
interaction between the faculty and IBSC members helped in clarifying the roles
and responsibilities of IBSC members in particular, the DBT nominees.
Keeping in view the feedback received in these
consultations, this handbook has been prepared by BCIL in consultation with DBT
for members and DBT nominees of IBSCs covering the composition, functions, role
in approval and a checklist for evaluating projects, which can be used on a
case-to-case basis. A CD containing
important biosafety regulations and guidelines for GMOs is also included in the
handbook for ready reference by the IBSC members and nominees of DBT.
As per Rules, 1989 notified by MoEF, an
IBSC is to be constituted by all organizations (including research
institutions) handling GMOs for research or production.
The IBSC shall have the following members:
(i)
Head of the Institution
or nominee
(ii)
3 or more scientists
engaged in rDNA work or molecular biology with an outside expert in the
relevant discipline.
(iii) A member with medical qualifications - Biosafety
Officer (in case of work with pathogenic agents/large scale use).
(iv)
A nominee of DBT.
Every institution setting up an IBSC
needs to identify the members as detailed above. The request has to be submitted to DBT along with the list and
background of the proposed members. DBT
then identifies an appropriate nominee and communicates the same to IBSC
chairman.
If research is conducted on organisms that require special containment conditions (Biosafety Level 3 or 4) or if large-scale microbial research is conducted, a Biological Safety Officer must be appointed. This person is also a member of the IBSC and acts as a technical liaison between researchers and the IBSCs. His responsibilities include periodic inspection of facilities and protocols and development of emergency plan, as higher containment levels require more scrutiny.
Each IBSC has to meet at least twice a
year to review the status of rDNA projects in the institution. It is important that the Chairman and Member
Secretary ensure that regular meetings take place. More than two meetings may be held as per requirement of the
projects. The IBSC members are expected to look into the following during the
meetings:
i.
Action taken on the decisions of earlier IBSC meetings.
ii.
Characterization of
work and approval as per risk category.
iii.
Evaluation of projects and direction to submission for appropriate
agencies for approvals
iv.
Inspection of
containment facilities and greenhouses etc.
v.
Review the medical
reports of employees
vi.
Maintaining procedures
and other approval requirements.
IBSC has to furnish half yearly reports
on the ongoing projects in the organization to RCGM regarding the observance of
the safety guidelines including accidents, risks and deviations, if any. The
prescribed formats for information on projects to IBSC/RCGM and half yearly
reports to be submitted to RCGM are given in Annexes I to IV.
The role of IBSCs assumes major
importance in the regulatory framework since it is a Statutory Committee that operates
from the premises of the institution and hence is in a position to conduct
onsite evaluation, assessment and monitoring of adherence to the biosafety
guidelines. The decisions taken by the next higher committee
i.e., Review Committee on Genetic Manipulation (RCGM), which operates from DBT
are based on the applications submitted by the investigators with the approval
of IBSC. Therefore, it is pertinent
that the members of the IBSCs and DBT nominees to the IBSCs have expertise in
evaluation, assessment and monitoring of projects as per the rDNA guidelines.
The functions in the IBSC of the head of
the organization, members, DBT nominees, and Principal Investigator are
explained below:
The head of
the institution, who is also the chairman of IBSC has the responsibility to
ensure that
·
the biosafety
guidelines are followed in his institution.
·
regular
meetings of IBSC are held to review recombinant research projects in the
institution.
·
open discussion takes place amongst the members in the
meetings and the views of external members as well DBT nominee recorded.
·
the facilities
at the institution are sufficient to meet the containment levels stipulated for
rDNA products and processes.
The main functions of IBSC members as
defined in the rDNA Safety Guidelines by DBT are as follows:
i)
Review and clearance of
project proposals falling under restricted category, which fulfill the
requirements under the guidelines.
ii)
Tailoring biosafety
programme as per the level of risk assessed.
iii)
Training of personnel
on biosafety.
iv)
Instituting a
health-monitoring programme for laboratory personnel.
v)
Adopting emergency
plans.
An indicative list of functions falling
under each of the above heads is given below:
i)
Review and clearance
of project proposals falling under restricted category and meet the
requirements under the guidelines:
·
Review biosafety
aspects of all projects submitted to IBSC.
·
Review categorization
of projects as per appropriate risk category.
·
Timely action as per
biosafety guidelines i.e. to be noted, approved for initiating work or
forwarded to RCGM.
·
Focus on scientific
details and containment facilities but corresponding review also for the
organization set up, status of other approvals required, and socioeconomic
aspects of rDNA projects.
ii)
Tailoring biosafety
programme to the level of risk assessment:
·
Risk assessment of the
projects by examining the intentional and unintentional consequences of the
specific modifications being targeted through genetic engineering.
·
Confirm assignment of
the appropriate containment level for the proposed work based on independent
assessment. The proper containment level for the unmodified organism should be
considered first followed by whether the proposed modified organism would leave
the level higher, lower or unchanged.
·
Review compliance with
the biosafety guidelines by evaluating facilities, procedures and the expertise
of personnel involved in the research projects.
iii)
Training of
personnel on biosafety:
·
To oversee that
procedures are in place in the organization for training of all staff and
students working with GMOs appropriate for the risk category under which the
experiments are planned/conducted. Emphasis on the procedures to be preferably
documented including specific responsibility and accountability for persons
within the organization.
·
To ensure that training
includes laboratory work practices for dealing with GMOs, awareness about
relevant rules and regulations and specific modules for persons dealing with
high-risk experiments. Training manual and other information to be provided to
new staff/students in regular orientation programs.
·
To ensure that the
record of all training provided is maintained by the organization for review as
well as onward submission to RCGM if required.
iv)
Instituting
health-monitoring programme for laboratory personnel:
·
To establish and
maintain a health surveillance programme involved in connection with the
individual rDNA projects, particularly those requiring higher containment
levels.
·
To review the complete
medical check-up of personnel working in projects involving work with GMOs as
required prior to starting such projects.
·
To review the follow up
medical checkups including pathological tests done periodically, at least
annually for scientific workers involved in such projects.
·
To ensure that medical
records are accessible to the RCGM.
v)
Adopting emergency
plans:
·
To review the emergency
plan proposed by the Principal Investigator for responding to an accidental release
and those adopted to meet any exigencies.
·
Copies of site
emergency plan to be submitted to RCGM, GEAC, State Biotechnology Coordination
Committee (SBCC) or District Level Committee (DLC) as the case may be.
Each IBSC has a nominee from DBT who
oversees the activities to ensure that safety aspects are being fully adhered
by the organization. The DBT nominee
serves as the link between the department and the respective IBSC. In addition to the responsibilities as an
IBSC member, the duty of the DBT nominee is to ensure that:
·
The committee has been
constituted as per the norms of the guidelines.
·
The Recombinant DNA
Safety Guidelines are strictly followed in the institution.
·
The IBSC meets
regularly, at least twice in a year to review the ongoing activities and
provides half yearly reports to RCGM/DBT in the prescribed performa.
·
All the activities are
within the purview of the guidelines and in the knowledge of RCGM/DBT.
·
The DBT nominee is
expected to guide the IBSC on biosafety issues.
All recombinant research projects carried out by an organization have a Principal Investigator (PI) and it is the duty of the PI to apprise the IBSC about the nature of the experiments being carried out. Depending upon the risk category, the PI has to inform the IBSC, seek permission of IBSC before starting the experiments or seek permission of the RCGM through its IBSC.
The PI is
primarily responsible for ensuring compliance with biosafety standards. The PI
functions as a project manager as well as a researcher, communicating with the
IBSC and bearing responsibility for training and supervising personnel. Based
on the nature of the GMO, the PI determines the proper containment level for
the project and, in accordance with the DBT Guidelines, develops the necessary
experimental protocols. This information is then submitted to IBSC for review.
The responsibilities of PI to IBSC are summarized below:
·
to make an
initial determination of the required levels of physical and biological
containment in accordance with the DBT guidelines.
·
to submit the
initial research protocol and any subsequent changes (such as changes in the
source of DNA or host vector system) to the IBSC for review and approval.
·
to ensure that
no work is initiated until the research project has been approved by the IBSC
and has met all requirements of DBT guidelines.
·
remain in
communication with the IBSC throughout the conduct of the project.
·
To ensure the
safe conduct of the rDNA experiments in his laboratory.
·
To make
available the protocols that describe the potential biohazards and the
precautions to be taken to all laboratory staff.
·
To instruct
laboratory staff about the practices and techniques required to ensure safety,
and the procedures for dealing with accidents including the reasons and
provisions for any precautionary medical practices advised or requested (e.g.
vaccinations or serum collection).
·
To supervise
the performance of the laboratory staff to ensure that the required safety
practices and techniques are employed.
·
To undertake
corrective measures promptly for any work errors and conditions that may result
in the release of recombinant DNA materials.
The
rDNA activities within an organization could be broadly categorized into
research, large-scale experiments/production/field release and import and
shipment. The role of IBSC in each of
these activities is explained below:
IBSC
has to review all recombinant research carried out by an organization. The rDNA Safety Guidelines of DBT stipulate
three categories of research activities i.e. Category I, II and III with
increasing level of containment requirements. Category I experiments involving
self cloning, using strains and also inter species cloning belonging to
organism in the same exchanger group etc. and are exempt for the purpose of
intimation and approval. Category II experiments falling under containment
levels II, III and IV, large scale use of recombinants made of self cloning in
systems belonging to exempt category etc. require prior intimation to
IBSC. Category III experiments
involving toxin gene cloning, cloning of genes for vaccine production, use of
infectious animals and plant viruses, self fusion experiments, field testing
and release etc. require review and approval of IBSC before commencement. Depending upon the category of experiments,
IBSC can simply note the information provided by PI, give permission before
start of the experiments or forward it to RCGM for approval.
The
categories of genetic engineering experiments on plants have been notified
specifically under the “Revised Guidelines for Research in Transgenic Plant,
1998” by DBT.
In this categorization, routine recombinant DNA experiments fall in Category I
and need only intimation to the IBSC in the prescribed performa. Category II include lab and
greenhouse/nethouse experiments in contained environment where defined DNA
fragments that are non pathogenic to human and animals are used for genetic
transformation of plants. Permission
for performing Category II experiments is provided by IBSC but the decision of
the IBSC needs to be intimated to the RCGM before execution of the experiment
and RCGM would put this information on record.
Category III pertains to high risk experiments where the escape of
transgenic traits into the open environment could cause significant alterations
in the biosphere, the ecosystem, the plants and animals by dispersing new
genetic traits, the effects of which cannot be judged precisely. All experiments conducted in greenhouse and
open field conditions not belonging to the above Category II types, would fall
under Category III risks. Such experiments could be conducted only after
clearance from RCGM and notified by the Department of Biotechnology.
Different
levels of containment have been prescribed for different categories of rDNA
experiments in the guidelines. IBSC
should 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 subject to
inspection on accountability.
Although the approval for small scale
field trials fall under the purview of RCGM and approval for large-scale trials
and production needs to be taken from GEAC, as per the Rules 1989, in all these
cases, IBSC has an extremely important role in terms of verifying the
information being forwarded to RCGM and GEAC in terms of physical containment
conditions, categorization in terms of risk assessment etc. being the statutory
body functioning on the premises of the institution. Both RCGM and GEAC depend
on the review of the IBSC on the submissions made.
IBSC has to recommend emergency plan in
case of large-scale operations, as and when required, which would be then
approved by competent authorities.
Emergency plan shall include methods and procedures for handling large
losses of cultures and organisms.
The interstate shipment of indigenous
etiological agents, diagnostic specimens and biological products need clearance
of IBSC and is subject to appropriate packaging, labeling and shipping
requirements.
The import 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
use are issued by RCGM while large scale imports for industrial use are regulated
by GEAC. In case of plants, the import
is routed through the Director, National Bureau of Plant Genetic Resources on
the basis of the import permit issued by the DBT, based on recommendations of
the RCGM. However, all these proposals need to be submitted by the PIs through
their IBSCs.
The aspects to be reviewed by IBSC in evaluating rDNA activities broadly include scientific considerations and availability of appropriate facilities. In addition, DBT nominee is also expected to review the organizational set up, facilities, and status of other approvals required/obtained, IPR and socioeconomic aspects of rDNA research projects on a case-to-case basis.
A detailed checklist is presented here to assist IBSC members in
reviewing the research proposals from investigators. It may be noted that this list is indicative. Specific additions/deletions would need to
be made to suit the requirements of each project on a case-to-case basis.
Risk assessment is a scientific process that makes use of the best up-to-date scientific knowledge and experience. Although details of risk assessment may vary from case to case, there are some logical steps that need to be followed. Table 1 gives the information required for risk assessment of a GMO.
Particulars
|
Information required |
|
Molecular
Biology Details Characteristics of donor organisms Characteristics
of host/ recipient organisms Characteristics of gene construct Characteristics of vector and method of transformation Characteristics of transformed/modified organism |
·
Origin/source and
taxonomic classification ·
Genome characteristics
and prior history for genetic manipulation ·
Nature of
pathogenicity and virulence, infectivity or toxicity, its host range and
stability of these traits ·
Techniques for
identification of donor organism ·
Origin/source and
taxonomic classification ·
Genome
characteristics and prior history for genetic manipulation,
cultivation/growth and safe use ·
Information on
reproductive cycle (asexual/sexual), its growth and survival ·
Nature of pathogenicity
and virulence, infectivity or toxicity, its host range and stability of these
traits ·
Degree of relatedness
including evidence of exchange of genetic material between donor and
recipient organisms or any other organisms ·
Origin ·
Physical map (including
coding regions, promoters and enhancers, marker genes, antisense genes) ·
Nucleotide sequence
of intended insert and its function/s ·
Nature, source and
function of vector ·
Method of
transformation and detailed description of transfer method ·
Selection method for
transformants ·
Type of insertion
(complete or partial) and number of integration sites ·
Confirming integrity
and fidelity of the insert ·
Determining
production of fusion protein and its location in recipient organism ·
Genotypic characters
including characterization of site of modification of recipient genome,
regulation and stability of inserted DNA, frequency of mobilization of
inserted DNA and/ or genetic transfer capability ·
Phenotypic characters
including taxonomic characterization, colonization potential, antibiotic
resistance, infectivity, production of toxins, allergens, antinutrients or
any other metabolites and its host range ·
Expression and
properties of the gene product including copies /number of new gene(s), rate
and level of expression, activity of expressed protein, allergenic/toxic
hazards of the product |
|
Human Health Considerations Toxicity
Allergenicity Nutritional
analysis |
|
|
Environmental Considerations Application
of the modified organism in the environment Survival, multiplication and
dissemination of the GMO in the large scale facilities/field/environment Characteristics affecting
survival, multiplication and dissemination Interactions
of modified organisms with biological systems (target and non-target
populations) Stability of the GMO in terms of genetic traits Routes of dissemination |
·
Geographical location
site ·
Containment and
decontamination ·
Introduction
protocols including quantity and frequency of application ·
Methods of site
disturbance or cultivation ·
Methods for
monitoring applications ·
Contingency plans ·
Treatment procedure
of site at the completion of application ·
Other including
transport arrangements, post-release monitoring of the site, impact of GMO on
weed formation
·
Known and predicted
habitats of the GMO ·
Description of the
target ecosystems and of ecosystems to which GMO could be disseminated ·
Identification and description
of target organisms ·
Anticipated mechanism
and result of interaction between the modified plant and the target
organism(s) ·
Identification and
description of non-target organism(s) which might be exposed
·
Routes of
dissemination, physical or biological ·
Known or potential
modes of interaction, including inhalation, ingestion, surface contact,
burrowing and injection |
|
Characteristics
of the purified products from GMOs (e.g.: recombinant therapeutics) |
|
8.1.1 Molecular
biology details:
Recombinant
DNA technology basically uses three components for manipulation i.e. the
selected gene from the donor organisms, the vector used for transfer of the
gene and the host organisms. Therefore,
the first step in risk assessment is to examine these three entities, followed
by the modified organism and the resultant gene products.
i.
Characteristics of the donor organisms:
If the donor organism is merely used as a source of well-characterized DNA for a selectable phenotype or a promoter or other control sequence, the characteristics of the donor are not very important to the risk assessment. If, however, the insert contain genes which are biologically active, producing toxins or virulence factors, then information from the donor organism is extremely important and of consequence. The construction of cDNA or genomic libraries helps in consideration of all the possible hazards associated with the donor organism.
Although, the characteristics of the donor organism are of less relevance to the risk assessment than those of the host, the hazard group selected would be generally higher of the two within which the host and donor fall.
ii.
Characteristics of the host/recipient
organisms:
A thorough knowledge of the host or recipient
organism is extremely important in assessment of the risks of the GMOs
particularly keeping in view the concept of substantial equivalence as a
starting point. The identity of the host must be established and the taxonomy
well understood. There should be adequate and documented experience of the safe
use of the host organism. The characterization of the host provides the
starting point for the risk assessment. The assumption that is generally taken
is that, the level of risk associated with the modified organism is at least as
great as that of the host organism (until proved otherwise).
In case of microorganisms, the pathogenicity of the organism is extremely important for the risk assessment and subsequent categorization. The host must be evaluated to determine that it is not pathogenic. Infection by a microorganism followed by disease depends on its ability to multiply in the host and on the host's ability to resist or control the infection. The microorganisms have been in categorized based on infectivity towards humans into four groups out of which the first group is that of non-pathogens (Table 2). This categorization is generally applicable only for the assessment of containment requirements as greater containment is required to control the organism in the higher hazard groups to ensure that the organism do not infect those working with it.
Table 2: Categorization of microorganisms based on
pathogenicity
|
Hazard
Group 1 |
Organisms
that are most unlikely to cause human disease |
|
Hazard
Group 2 |
Organisms
capable of causing human disease and which 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
Group 3 |
|
|
Hazard
Group 4 |
Organisms
that cause severe human disease and are a 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 |
The details of microorganisms falling into each category are given in the Recombinant DNA Biosafety Guidelines, 1990.
Some organisms have been used in rDNA technology experiments
frequently and their characteristics have been described in detail e.g. E. coli
or Saccharomyces cerevisae are organisms about which a great deal is
known. Further no pathogenic strains of bakers' or brewers' yeast have ever
been observed. This type of familiarity allows some confidence in attempting to
identify risks associated with their modification. Some strains for example, E.
coli K12 has been disabled to remove some of the factors that might be
associated with pathogenicity (wild type E. coli is a Hazard Group 2
pathogen). The factors which have been lost include the cell-surface K antigen,
part of the LPS side chain, the adherence factor (fimbriae) that enable
adherence to epithelial cells of human gut, resistance to lysis by complement and
some resistance to phagocytosis. This variant of E. coli is a common
host organism for genetic modifications within the laboratory.
In case of crops being used as the hosts for genetic
manipulation, additional factors such as potential invasiveness of the
species need to be considered. Plant
species have different geographical ranges and estimates of invasiveness may
vary in different regions. Crops can be
divided broadly into six categories in accordance with their invasive
potential:
i.
Crops that have no
compatible relatives, carry few weediness traits (less than 40 percent), and do
not persist in natural environments.
ii.
Crops that have no
compatible relatives, carry intermediate numbers of weediness traits, rarely escape,
and do not persist in natural environments.
iii.
Crops that have no
compatible wild relatives, carry many weediness traits, and can escape and
persist in natural environments.
iv.
Crops that have
compatible relatives, carry few weediness traits, and can escape but do not
persist in natural environments; their compatible relatives also carry few
weediness traits and dot not aggressively spread
v.
Crops that have
compatible relatives, carry intermediate numbers of weediness traits, and can
escape but do not persist in natural environments; their compatible relatives
also carry few weediness traits and do not aggressively spread.
vi.
Crops that have
compatible wild relatives, carry many weediness traits, and can escape and
persist in natural environments; their compatible relatives also carry many
weediness traits and aggressively spread.
The relative risk of using a transgenic crop will
increase with the degree of invasiveness.
The relatedness between the host and the
donor organisms is also important in the risk assessment particularly with
respect to exchange of genetic material between them as well as with other
organisms.
iii.
Characteristics of the insert/ gene construct:
The properties of the insert are extremely important in risk assessment of GMOs. For example if the information encodes a toxic gene product, or one which is known to be likely to modify the pathogenicity of the organism into which it is inserted, the greater the risk. However, if the gene product is non-toxic and is not one which may pose a risk to the people working with the organism in containment, the risk management will largely be based on the pathogenicity of the host organism. In case of plants, the transgenes for herbicides and pest resistance need more careful scrutiny as compared to the ones that are selectively neutral in the natural environment.
Individual components used in the preparation of the construct i.e. promoters, enhancers and marker genes also need to the carefully reviewed.
iv.
Characteristics of the vector and method of
transformation:
The vector has to be characterized both for its own potential for pathogenicity and for its ability to transfer the insert to organisms other than the intended horizontal transfer. The function of the genetic material on the vector should be known as this would ensure that the vector is free from sequences that could be harmful to humans or the environment. The vector should be limited in size as much as possible to the genetic sequence required to perform the intended functions. This decreases the probability of introduction and expression of cryptic functions or the acquisition of unwanted traits. The presence of genes coding for antibiotic resistance might be of concern, although, for most of the vectors the antibiotic resistance is already common in the environment.
The methods of transformation used for introducing the required gene should be considered for the risk assessment of the modified organism. For example, in case of plants, the two principle methods of transformation that are widely used are the Agrobacterium mediated transformation and particle bombardment. Whereas Agrobacterium mediated transformations result in a low transgene copy number, minimal rearrangement and higher transformation efficiency, particle bombardment causes extensive rearrangements to transformed sequences.
v.
Characteristics
of the modified organism:
Molecular characterization of the GMO is used to provide information about the composition and integrity of inserted DNA, the number of copies of inserted DNA, the number of sites of insertion and the level expression of novel proteins over time and in different tissues in case of plants and animals. Molecular characterization can provide useful information but cannot by itself answer all questions on risk assessment and safety of GMOs.
The inheritance and stability of each introduced trait i.e. functional in the modified organism must be determined. For each novel trait the pattern and stability of inheritance must be demonstrated as well as the level of expression of the trait by estimation of protein and its analysis. If the new trait is one that does not result in the expression of new or modified protein then its inheritance will have to be determined by examining the DNA insert directly or by measuring RNA transcript production.
The first presumption for risk assessment
is that the modified organism is at least as hazardous as the host. For
example, work with modified haemolytic streptococci will proceed in the
laboratory in a similar way as with other streptococci of this type and of
known pathogenicity. However, more precautions are normally required for
modified organisms as introduced external DNA might increase the hazard usually
attached to these haemolytic streptococci. Formally such potential increase of
the hazard is expressed by classification of the manipulated strain in higher
risk category. The formulation "might increase" is important since it
reflects the lack of familiarity with the new strain. In some cases it may be
observed that the opposite happens i.e. the new strain will be less invasive,
the haemolysis less expressed. In short - the strain will represent less hazard
to human health. Nevertheless, the new strain has to be treated as more
dangerous until confirmed otherwise.
Risks associated with a GMO can be assessed by considering three factors i.e. access, damage and expression. Access is a measure of the probability that a modified organism, or the DNA contained within it, will be able to enter the human body and survive there or escape into the environment as the case may be. It is a function of both host and vector. The properties of the vector, particularly mobilization functions need to be taken into account. Expression and damage are usually associated with the insert and the gene product.
Expression is a measure of the anticipated or known
level of expression of the inserted DNA. If the 'gene' inserted is intended to
be expressed at a high level, for example, by deliberate in-frame insertion
down-stream of a strong promoter, expression is likely to be high. If the
insert is simply there to allow probes to detect the DNA, and is
non-expressible DNA, i.e. with no foreseeable biological effect or gene containing
introns, which the host is incapable of processing, then the expression factor
will be low. Examination of the modified organism determines the actual
expression, which may be higher or lower than expected.
Damage is a measure of the likelihood of
harm being caused to a person by exposure to the GMO, and is independent of
either expression or access. It is associated with the known or suspected
biological activity of the DNA or of the gene product. The activity of the
organism, which results in any toxic, allergenic or pathogenic effect need be
taken into account within this parameter. It may be that the biological
activity of a protein is dependent on the host cell system in which it is
expressed. An oncogene expressed in a bacterium will have no discernible
effect, but when it is present in a human cell, problems may arise. The full
biological function of many gene products requires post-translational
modification, which will not occur within a bacterial cell normally. The
potential biological activity of the gene product should be considered in the
context of where and how it has been expressed and the effect on its structure
and activity of the mode of manufacture.
Once an estimate of each of these parameters has been
made, they may be combined. The result provides a qualitative measure of the
risk, and allows a containment level to be assigned for the use of the
organism.
The
categorization scheme based on risk assessment has been given in Recombinant
DNA Guidelines 1990, which should be referred to for evaluating the containment
requirements as well as approvals to be taken.
8.1.2
Human
health considerations:
Impact on human health is studied by analyzing the modified organism for the risks of toxigenicity, allergenicity, pathogenicity ,teratogenicity etc. as relevant in the particular situation. Assessment procedures and criteria vary in each case of genetic modification carried out in microorganisms, plants, animals etc. and products thereof, some of which are briefly explained below:
i.
Toxicity studies:
The
main toxicological assessment of a GMO deal with the protein expression studies
of inserted gene(s). Another concern is
the expression of novel proteins in host organisms due to genetic modification and
resulting changes in the metabolism, if any. In vitro and in vivo studies
are needed to assess the toxicity levels of GMOs and products thereof. The standard toxicology methods are often
well documented in the scientific and technical literature and the appropriate
protocols can be drawn for each GMO.
In transgenic plant tissues, the concentration of novel protein
expressed can be very low, often much less than 0.1% on a dry weight basis.
Studies, such as acute toxicity testing, which require relatively large amounts
of material are often not feasible using the protein purified from plant
tissue. Instead, these studies normally make use of protein purified from
bacterial expression systems. In such cases, it is necessary to demonstrate the
functional equivalence (i.e., equivalence of physicochemical properties and
biological activities) of proteins purified from the two sources. When
equivalence is demonstrated based on serological cross-reactivity, it is
important to use antisera (either polyclonal or monoclonal) that have been well
characterized with respect to their specificity.
ii.
Allergenicity:
These
risks are more difficult to determine except in simple cases where the
transgenes come from a species that is known to involve a risk of allergic reactions
or even codes for an already identified allergen. If not, the assessment may be based on the structural
similarities between the product of the transgene and known allergens and on
the residual levels of the proteins coded by the transgene in the product for
use/consumption. Databanks for
potentially allergic peptides are available that facilitate these studies. Detailed protocols have been defined in the
guidelines which may be referred to while evaluating the proposals.
As
an example, in genetically modified plants the common criteria to make
decisions regarding allergenicity can include:
a.
whether the source of
genetic material is known to contain allergens.
b.
assessment of amino
acid sequence of allergens.
c.
immunoreactivity
assessment.
d.
effect of pH and/or
digestion since most allergens are resistant to gastric acidity and to
digestive proteases
e.
heat or processing
stability studies
iii.
Nutritional analysis:
Nutritional
analysis is necessary for GM food crops being developed. Unintended changes in level of nutrients and
expression of other biochemicals can occur in many ways including through
insertion of genetic material. Food safety assessments should consider the
potential for any charge in nutritional composition. For genetically engineered
plants aiming at altered nutritional value, the nutritional evaluation should
demonstrate that there has been non intentional charges in the levels of key
nutrients, natural toxicants or anti nutrients or the bioavailability of
nutrients.
iv.
Environmental
considerations:
In addition to the effect of inserted gene(s) and
their impact on genotype and phenotype of a modified organism, it is important
to study the proliferation of the GMO in the environment and the effect on its
equilibrium.
Environmental
risk assessment of GMOs must be undertaken on a case to case basis and there
can be no single method or model to follow. Broader issues include the
potential adverse effects, likelihood of these risks becoming a reality,
consideration of risk management strategies and assessment of overall potential
environmental impact.
Possible
adverse effects include outcrossing between GM organisms and pathogens,negative
effect on population of non target organisms, including indirect effects on
population levels of predators, competitors, herbivores, symbionts, parasites
and pathogens.
Identification
of any potential adverse effect is followed by a stage in which an estimation
is made of the likelihood that the identified potential adverse effect will actually
occur. It is important to estimate the chances of each of potential effect for
assessment purposes.
The
likelihood of certain potential adverse effects occurring can be influenced by
characteristics of the size and scale of application in addition to those of
inserted transgene and the recipient organism.
A precautionary approach is useful in assessment of ecological effects.
In general, biosafety begins with ensuring the workplace whether it is a laboratory, fermentation plant or open fields, safe for the working staff, the general population and finally, the environment by proper containment.
Containment covers both the research stage, when modifications are made, development work in the laboratory, greenhouse or growth room, manufacturing units where GMOs are used for production and open fields where they are released. When a new research project is initiated, it involves the modification of organisms within a laboratory under very controlled conditions. The risks are perceived only to those working in the laboratory and containment conditions are devised to ensure that the organism would not escape into the environment, or if it should, it would have been so design not to survive in the open. At this stage, the associated risks are mainly to the human health. However, when the GMOs are used in an industrial or commercial environment, or in open cultivation, the volume of material is considerably larger and the individuals working with GMOs may be less knowledgeable or competent at handling the situation. This implies that there is possibility of accidental escape in a volume large enough for the GMO to survive and persist in the open environment. There is also a risk of accidental release where the waste from industrial unit/fields is not as carefully monitored as in the laboratory. Therefore, the containment requirements in these cases would take into account both impact on human health and possible environmental effects.
The containment could be physical, where there are real barriers to prevent escape or biological where the organism is designed not to be able to survive in any environment other than that of the laboratory. The containment facilities and biosafety practices have been defined in detail in “Recombinant DNA Safety Guidelines, 1990” of DBT. In brief, the basic laboratory guidelines have been detailed that are fundamental to all classes of risk groups followed by modifications for work with more dangerous pathogens. For more details, the most reliable reference is Laboratory Biosafety Manual of the World Health Organization is available at its website. A summary of recommended biosafety levels for infectious agents is given in Table 3:
Table 3: Summary of
recommended Biosafety Levels for Infectious Agents
|
Biosafety Level |
Practice and Techniques |
Safety |
Facilities |
|
1. |
Standard microbiological practices |
Non primary containment provided by
adherence to standard laboratory practices |
Basic |
|
2. |
Level 1 practices plus laboratory coats;
decontamination of all infectious wastes limited access; protective gloves
and biohazard warning signs as indicated |
Partial containment equipment (i.e. Class I
or II Biological Safety Cabinets) used to conduct mechanical and manipulative
procedures that have aerosol potential that may increase the risk of exposure
to personnel |
Basic. |
|
3. |
Level 2 practice plus special laboratory
clothing, controlled access |
Partial containment equipment used for all manipulations
of infectious material |
Containment |
|
4. |
Level 3 practices plus entrance through
change room where street clothing is removed and laboratory clothing is put
on shower on exit, all wastes are decontaminated on exit from the facility |
Maximum containment equipment (i.e. class
III biological safety cabinet or partial containment equipment in combination
with full body air supplied, positive pressure personnel suit used for all
procedures and activities |
Maximum containment |
It may be noted that effective physical containment of bacteria, viruses and other microbes can be extremely difficult because they cannot be seen and once disbursed cannot be recovered. Biological measures often provide better containment options in these cases. Using biological and physical containment measures in concert offers advantages to achieve a specified level of containment. It may also reduce the physical requirements to those of the next lower biosafety level. For example, an experiment design to evaluate tomato plants genetically engineered for resistance to tomato spotted wilt virus involves three organisms i.e. tomatoes, the virus and thrips, the insect vector that transmits the virus. Whereas physical containment would be provided by a greenhouse with antivirus screening or by conducting the experiment in insect proof cages within the greenhouse, biological containment could be added by removing alternate host plants for the virus both in and outside of the greenhouse and by applying stringent insect control measures in the surrounding area.
A detailed
checklist for according approval to a laboratory for carrying out recombinant
DNA technology work has been given in “Recombinant DNA Safety Guidelines, 1990”
of DBT. In addition to these
guidelines, some of the key points as reviewed from WHO guidelines are detailed
below:
i.
Premises and lab:
Ř Appropriate containment: code of practice; lab design and facilities; health and medical surveillance; specification for gene technology lab; specification for large scale operations
Ř Prevention against entry of pests (air pressure, exhaust air, input air)
Ř Provisions for emergency
Ř Provisions for storage and disposal: In process material; starting material; finished product; infected material/rejected
Ř Cleanliness
and hygiene
Ř Repair
facilities
ii.
Equipment:
Ř Adequacy of equipment: appropriate design; set up and
maintenance
Ř Standard Operating Procedures (SOP): validation of
all equipment; calibration of all instruments; investigating recording all
deviations and expertise
Ř Automated equipment: computer controlled system;
back-up file maintenance and hard copy systems
iii.
Animal
facilities:
Ř 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
Ř Pest control system; facilities for waste, carcass;
cleaning, sterilization and maintenance of supplies and equipment (animal
cages, racks)
iv.
Environment:
Ř SOPs to minimize contamination; monitoring frequency;
methods for viable counts in air, water, surface and non viable particulates in
air.
The checklist suggested above is for
standard requirements of a laboratory carrying out work in recombinant DNA
technology. Appropriate reviews need to be undertaken based on the type of
organisms used and the experiments planned.
8.3.1
Status of
the organization:
It is important to make a note of the status of the
organization vis-ŕ-vis its activities to get an idea on the capabilities to
undertake the proposed research activities. In case of industry, the activities
of the company or its associates in the areas other than biotechnology should
also be reviewed to give an overall idea of organization’s standing and
approach. In case of institutions, any affiliation to apex bodies such as CSIR,
UGC, ICMR and ICAR should be noted. If it is an independent research institute,
credentials should also be reviewed.
The observations with regard to the status of organization help in
reviewing long-term accountability to meet statutory requirements. The indicative points are given below:
i. Constitution - university departments/Industry/ research institution/ deemed university/ private research laboratory/contract research organizations
ii. Affiliation – national bodies/CSIR/UGC/ICMR/ICAR/ major industrial groups
iii. Braches – subsidiaries organization
iv. Major activities
v. Year of establishment
vi. Year of initiating rDNA activities
vii. Year of setting up IBSC
8.3.2 Organizational
structure:
Information regarding organizational framework such as levels of authority, chain of command, product range etc. needs to be inspected. It is necessary to ensure that supervision at various levels by senior scientists is built in the organization structure. The number of specialists having appropriate qualifications and skills should be looked at as to whether they are in line with the complexity of the proposed projects. Necessary provisions against unauthorized entry and staff movement in restricted areas of the organization should also be in built to ensure the compliance of the assigned risk category.
8.3.3
Source of
funding:
The sources of funding of
research projects in biotechnology should also be specified by the PI and
reviewed by IBSC. The funding could be
from the government departments or apex bodies such as DBT, DST, UGC, CSIR, ICMR
etc., industry, NGO/donor organizations. This helps in understanding the
objective of the proposed research in terms of economic implications and
benefits to the country. The information may be collected as follows:
i.
Estimated project cost
ii.
Estimated time for
development
iii.
Stage of development –
basic research; lab scale development; large scale manufacture; marketing
iv.
Sources of funds for
development - In-house; State/Central Government (such as DBT, DSIR, MoH,
MoEF); International organizations (UNDP, UNIDO, FAO etc.) Industry; National
and International NGOs/Donors etc.
8.3.4
Status of
other approvals required:
Apart from the approval of IBSC, the rDNA activities
under consideration may require approvals from other regulatory agencies such
as Drug Control Authorities, Animal Ethics Committee and Plant Quarantine
Authorities. Further, the status of
accreditation by organizations such as DSIR may also be reviewed. When the facilities have been set up as per
the GMP/GLP specifications and have certification from reputed agencies such as
WHO, ISO etc., IBSC would be able to recommend conducting experiments in higher
risk categories.
Detailed documentation also contributes to the building up of familiarity, which in future may lead to amendment of the risk assessment and an assignment of a lower level of containment.
8.3.6 Training/skill
requirements:
GMOs may be used in contained laboratories or pilot plants. Alternatively, they may be used in an industrial setting or open fields. It may be noted that normally a research and development laboratory will be working with organisms, which pose a greater threat to either the individuals working therein or to the environment than those organisms, which have been developed for large scale use. The majority or organisms used in industrial production/large scale used are well characterized. On the other hand at the development stage when the modified genes are inserted into organisms, the unpredictability of insertion site requires greater care than that taken at the production facility. Organisms used in the research laboratory may be pathogenic to humans and/or harmful to the environment. Experiments could involve organisms and/or inserts, which may be injurious to the health of the workers or to those who are incidentally in the laboratory. It is therefore extremely important that the research and laboratory staff are well trained in biosafety requirements.
8.3.7
Other
issues:
Although other issues like Intellectual Property Rights, socio economic benefits and ethical issues are distinct from biosafety assessments, they need to be looked into carefully for addressing the concerns of the public. IBSC should advise the PI to review the status of the project with respect to these considerations as well.