6.2 IBSC members
6.3 DBT Nominee
VII. Application to GEAC seeking approval under rules 8,9,10 & 11 of the Notification No. GSR 1037(E) dated 05.12.1989 issued by the Ministry of Environment and Forests under The Environment Protection Act, 1986, for transgenic plants certification
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.
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
· 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
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.
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
Practice and Techniques
Standard microbiological practices
Non primary containment provided by adherence to standard laboratory practices
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
Level 2 practice plus special laboratory clothing, controlled access
Partial containment equipment used for all manipulations of infectious material
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
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
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)
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.