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					                                              DRAFT FOR COMMENTS/COMPLIANCE

 GUIDANCE FOR INFORMATION/DATA GENERATION AND DOCUMENTATION
 FOR SAFETY ASSESSMENT OF REGULATED, GENETICALLY ENGINEERED
                         (GE) PLANTS


Safety assessment of a GE plant is the most important step in its development
process. The data to be generated for safety assessment of regulated, GE plant
involves research/experiments to be undertaken both under contained facilities as
well as confined field trials. These studies can be broadly categorized as:

I.     Studies that should be completed before Biosafety Research Level I (BRL-I)
       trials are undertaken
II.    Field studies that should be completed during BRL-I and/or Biosafety
       Research Level II (BRL-II) trials
III.   Non-field studies that should be completed in parallel to BRL-I and BRL-II.

The checklists provided herewith under each of the above category are provided to
assist the applicants (product developers), as they undertake research including the
confined field trials to address the food, feed and environmental safety of regulated
GE plants. It may be noted that the checklists provided here are indicative and the
applicants should read various regulations and guidelines issued by the Department
of Biotechnology (DBT) and the Ministry of Environment and Forests (MoEF) (list
placed at Annex-1) and may consult the regulatory agencies for any specific issues
regarding the data requirements on a case by case basis. The checklists may also
be used for compiling the data/preparing the dossiers to be submitted to regulatory
authorities for seeking approval at various stages of approval process. A flow chart
giving a broad list of studies is placed at Annex-2.


I.     STUDIES THAT SHOULD BE COMPLETED BEFORE BRLI TRIALS ARE
       UNDERTAKEN

       Before submission of an application to RCGM for permission to conduct BRL-I
       trials it is recommended that applicants will have completed the following:

       Checklist 1: Description of the Genetically Engineered Plant
         □ Identification of the crop (including the botanical and local names)
         □ Name/number of the transformation event(s)
         □ Pedigree map for each transformation event
         □ Purpose of the modification (introduced trait)

       Checklist 2: Description of the Biology of the Non-Transgenic Host Plant

       DBT and MoEF have prepared biology documents for some plant species that
       can be referenced by the applicant in lieu of compiling the information below.
       If a biology document is not available from the regulatory agencies i.e. DBT
       and MoEF, the applicant will have to prepare the same to address the
       information requirements outlined below. It is recommended that applicants
       consult with DBT/MoEF on the format and content of a biology document
       before its preparation begins.
                                          1
   □ Taxonomy, geographic origin and domestication of the plant
     o Taxonomy
     o Relatives of the species
     o Geographic origin (centre of origin)
     o Domestication
     o Germplasm diversity
   □ Reproductive biology
     o Growth and development
     o Floral biology
     o Pollination and Fertilization
     o Asexual Reproduction
     o Dissemination of Seed
     o Mating systems
   □ Naturally occurring crosses
     o Intra- and inter-specific crosses
     o Natural crossability
     o Inter-generic hybridization
     o Wild relatives in India
   □ Ecological Interactions
     o Gene flow
     o Volunteers and weediness
     o Potential for gene transfer to other plants
     o Free-living populations
   □ Human health considerations Any known endogenous toxins, allergens
     or anti-nutrients
   □ Cultivation in India
     o Climatic and soil types
     o Breeding objectives, milestones in breeding advances and
         challenges
     o Zonalization of varietal testing
     o Significant pests and pathogens of the plant species in India
     o Significant beneficial organisms associated with the plant species in
         India

Checklist 3: Description of the Genetic Modification(s)

   □ Information on the specific method used for the modification
   □ Description and characterization of all genetic material used to modify
     the plant, including the source (e.g., plant, microbial, viral, synthetic),
     identity and expected function in the plant
   □ Details of modifications to introduced, intermediate and recipient
     genetic material (e.g., changes in amino acid sequence that may affect
     expression of the expressed protein)
   □ A summary diagram of all genetic components of the vector, including
     coding regions, and non-coding sequences of known function and for
     each genetic component include:
     o A literature citation where these functional sequences are
         characterized.
     o Portion and size of the sequence inserted.
     o Location, order, and orientation in the vector.
     o Function of each component in the plant.

                                   2
            o Source (common, scientific and/or trade name and taxonomic
              classification of the donor organism) of each genetic element.
            o Information if the genetic component is responsible for disease or
              injury to plants or other organisms and is a known toxicant,
              allergen, pathogenicity factor, or irritant.
            o Information if the donor organism is responsible for any disease or
              injury to plants or other organisms, produces toxicants, allergens or
              irritants or whether closely related to organisms that do.
            o Information if there is a history of safe use of the donor organism or
              components thereof, if available.

      Checklist 4: Assessment of Possible Toxicity and Allergenicity
        □ Comparison of the amino acid sequence homology of the newly
           expressed protein and known protein toxins and anti-nutrients.
        □ Comparison of amino acid sequence homology of the newly expressed
           protein and known allergens.

      Checklist 5: Conformation of Inheritance of the New Trait(s) Over
      Multiple Generations
        □ Data to demonstrate the pattern and stability of inheritance of the new
            trait(s) over multiple generations in a manner consistent with the laws
            of Mendelian inheritance.
            o Data should be provided for at least five generations of the event.
            o Inheritance may be demonstrated through the use of serological
                techniques used to measure trait expression either qualitatively
                [e.g., Western immunoblotting, enzyme linked immunosorbent
                assay      (ELISA),   etc.]   or    quantitatively  (e.g.,    ELISA,
                radioimmunoassay, etc.) or through a validated functional assay
                (e.g., bioassay).
        □ If the new trait is one that does not result in the expression of a new or
            modified protein/biomolecule (e.g., transgenic plants containing
            inserted antisense sequences) then its stable inheritance will have to
            be determined by examining the DNA insert directly (e.g., by Southern
            blot or PCR) or by measuring RNA transcript production.




II.   FIELD STUDIES THAT SHOULD BE COMPLETED DURING BRL1 AND/OR
      BRLII TRIALS

      The studies in this section include both in-field experiments as well as
      studies that require plant material collected from field trials as in both
      cases design of the field trials must be given careful consideration. The
      applicant should carefully review the studies summarized below to identify
      when data should be collected over one or two crop seasons. It is
      recommended that whenever possible applicants try to complete these
      studies during the first two seasons of field trials.




                                         3
Checklist 6: Conformation of Expression Levels of New Protein(s)

   □ Quantify the level of expression of the gene product (e.g., protein)
     associated with each introduced trait:
     o Expression levels should be quantified in relevant plant tissues
        including: seed or grain; above ground vegetative tissue; roots;
        pollen, and any additional plant parts that may be used as livestock
        feed or for food.
     o Data should be collected from test plants produced in replicated
        field trials over the course of two crop seasons.
     o Each field trial should include at least two negative controls: the
        non-transformed parental line and at least one other non-
        transformed control variety/hybrid representative of varieties/hybrids
        of that plant species typically cultivated in the area (local varieties)
        where the trial is planted.
     o Data should be collected from a minimum of three trial site locations
        representative of the range of agro-ecosystems where the
        genetically engineered plant may be cultivated. More than three
        field trial sites may be required to ensure that the normal range of
        agro-ecosystems where that plant species is cultivated is
        adequately represented.

Checklist 7: Reproductive and Survival Biology
  □ Information and data about the reproductive and survival biology of the
     transformed plant and its non-transformed counterparts (see Table 1).
     o Data should be collected from test plants produced in replicated
         field trials over at least two crop seasons.
     o Each field trial should include at least two negative controls: the
         non-transformed parental line and at least one other non-
         transformed control variety/hybrid representative of varieties/hybrids
         of that plant species typically cultivated in the area where the trial is
         planted.
     o Data should be collected from a minimum of three trial site locations
         representative of the range of agro-ecosystems where the
         genetically engineered plant may be cultivated. More than three
         field trial sites may be required to ensure that the normal range of
         agro-ecosystems where that plant species is cultivated is
         adequately represented.




                                    4
   Table 11 Characteristics to be evaluated in studies of the reproductive and survival
                        biology of the genetically engineered plant
Characteristic                         Notes
Growth habit                           Record observations on changes in basic
                                       morphology of the plant including any
                                       abnormalities
Life cycle                             Describe if the introduced trait is anticipated to
                                       result in any change affecting whether the
                                       plant remains categorized as annual, biennial
                                       or perennial.
Plant height and biomass               To be recorded at regular intervals during the
                                       growing season.
Impact on pollinator species           This may be addressed through information
                                       on whether the same pollinator species have
                                       been observed in the field or if there have
                                       been changes in visiting pollinators (requires
                                       previous information on pollinators of the non-
                                       genetically engineered plant species)
Indicators of changes in weediness Describe if the introduced traits are likely to
potential or aggressiveness            affect dissemination of seed
                                       Evaluate in-field germination and dormancy of
                                       seed
                                       Measure:
                                              Number of flowers produced per plant
                                              Number of fruits produced per plant (or
                                               grain yield as appropriate)
                                              Pollen production, morphology and
                                               viability
                                              Time to maturity
                                              Number of flowering days
                                              Number of viable seeds produced per
                                               fruit
                                              Percentage of germinated seeds
                                               surviving to maturation
Biotic stresses                        Observations of susceptibility to pests and/or
                                       diseases commonly associated with the plant
                                       species (requires previous information on
                                       pollinators of the non-genetically engineered
                                       plant species).
Abiotic stresses                       Observations of responses to drought stress
                                       or nutrient deficiency or other stresses
                                       common to the plant species where
                                       applicable.




1
 On a case by case basis any additional studies may be required or some studies may not be
warranted based on the biology and phenotype of the genetically engineered plant or where the
applicant can justify the exclusion of a study using scientific rationale.
                                                 5
Checklist 8: Compositional Analyses of Key Components

In order to harvest the appropriate type of plant material(s) for compositional
analyses, the applicant should consider the following:

   □ Plant material for compositional analyses should be from test plants
     produced in replicated field trials over at least one crop season.
   □ Each field trial should include at least two negative controls: the non-
     transformed parental line and at least one other non-transformed
     control variety/hybrid representative of varieties/hybrids of that plant
     species typically cultivated in the area where the trial is planted.
   □ Plant material should be collected from a minimum of three trial site
     locations representative of the range of agro-ecosystems where the
     genetically engineered plant may be cultivated. More than three field
     trial sites may be required to ensure that the normal range of agro-
     ecosystems where that plant species is cultivated is adequately
     represented.
   □ Sampling of adequate number of plants and the methods of analysis
     need to be sufficiently sensitive and specific to detect variations in key
     components.

For all other parts of the GE plant and its conventional counterparts that may
be used as food or livestock feed, provide the following:

   □ Proximate composition e.g., ash, moisture content, crude protein,
     crude fat, crude carbohydrate;
   □ Content of true protein, non-protein nitrogenous material (e.g., nucleic
     acids and aminoglycosides), amino acid profile [unusual amino acids
     should be determined if their presence is suspected (e.g., d-amino
     acids from bacterial proteins)];
   □ Quantitative and qualitative composition of total lipids, i.e., saponifiable
     and nonsaponifiable components, complete fatty acid profile,
     phospholipids, sterols, cyclic fatty acids and known toxic fatty acids;
   □ Composition of the carbohydrate fraction e.g., sugars, starches, chitin,
     tannins, non-starch polysaccharides and lignin;
   □ Qualitative and quantitative composition of micronutrients, i.e.,
     significant vitamin and mineral analysis;
   □ Presence of naturally occurring or adventitious anti-nutritional factors
     e.g., phytates, trypsin inhibitors, etc.;
   □ Predictable secondary metabolites, physiologically active (bioactive)
     substances, other detected substances.




                                    6
        Checklist 9: Impact on Non-target Organisms

        In order to evaluate the potential impact of the genetically engineered plant on
        non-target organisms, applicants should undertake the following:

            □ Tier 1 studies: Laboratory-based, non-target organism studies using
              test diets incorporating concentrations of the target protein at, or
              above,      the     maximum       estimated     environmental    exposure.
                                                         2
              Representative non-target organisms typically used in Tier 1 testing
              are:
                  o Mammalian e.g., mouse
                  o Avian e.g., northern bobwhite quail (Colinus virginianus)
                  o Freshwater fish e.g., channel catfish (Ictalurus punctatus)
                  o Aquatic invertebrate e.g., Daphnia magna
                  o Non-target arthropods:
                          Honey bee larvae and adults (Apis mellifera)
                          Lady beetle (Hippodamia convergent)
                          Green lacewing (Chrysopa carnea)
                          Parasitic hymenopteran (Brachymeria intermedia)
                          Collembola
                          Earthworm (Lumbricus terrestris)
            □ Tier 2 studies: Field based studies that require the assessment of the
              actual abundance of non-target species under test and control
              conditions.
                  o Data should be collected from test plants produced in replicated
                     field trials over at least one crop seasons.
                  o Each field trial should include at least two negative controls: the
                     non-transformed parental line and at least one other non-
                     transformed        control     variety/hybrid   representative    of
                     varieties/hybrids of that plant species typically cultivated in the
                     area where the trial is planted.
                  o Data should be collected from a minimum of three trial site
                     locations representative of the range of agro-ecosystems where
                     the genetically engineered plant may be cultivated. More than
                     three field trial sites may be required to ensure that the normal
                     range of agro-ecosystems where that plant species is cultivated
                     is adequately represented.
                  o These studies should be designed to consider:
                          Threatened and endangered species in the area where
                              the crop is to be grown;
                          Beneficial organisms, including:
                                    Primary pollinators
                                    Predators
                                    Parasites
                                    Biological control organisms
                                    Soil microbes
                                    Other appropriate non-target organisms.


2
  The representative non-target organisms listed here are those recommended by the United States
Environmental Protection Agency. The determination of which non-target organisms may be adequately
representative of the Indian agro-ecosystem should be done in consultation with RCGM.
                                                   7
       Checklist 10: Livestock Feeding Performance Studies

       Livestock feeding trials as required on a case-by case basis. For more
       information please refer to the DBT publication “Protocols for Food and Feed
       Safety Assessment of GE Crops” available at http://igmoris.nic.in/.


III.   NON-FIELD STUDIES THAT SHOULD BE COMPLETED IN PARALLEL TO
       BRL-I AND BRL-II
       During the period that confined field trials will be undertaken, other non-field
       studies necessary to meet information and data requirements for the
       environmental risk assessment of GE plants should also commence.

       Checklist 11: Molecular Characterization of the Genetic Engineered
       Plant

       Describe:
         □ The gene product(s) (e.g. a protein or an untranslated RNA);
         □ The gene product(s)’ function;
         □ The phenotypic description of the new trait(s).

       Provide information about the DNA insertion(s) into the plant genome,
       including:
           □ Characterization and description of the inserted genetic material.
           □ Number of insertion sites.
           □ Organisation of the inserted genetic material at each insertion site
              including:
                  o Copy number
                  o Data to demonstrate if complete or partial copies were inserted
                  o Data to demonstrate if the arrangement of the genetic material
                     was conserved or if significant rearrangements have occurred
                     upon integration.
           □ Sequence data of the inserted material and of the flanking regions
              bordering the site of insertion.
           □ Identification of any open reading frames within the inserted DNA or
              created by the insertions with contiguous plant genomic DNA including
              those that could result in fusion proteins.

       Checklist 12: Assessment of Possible Toxicity (Proteins)

          □ Demonstrate the susceptibility of each newly expressed protein to
            pepsin digestion.
          □ Where a host other than the transgenic plant is used to produce
            sufficient quantities of the newly expressed protein for toxicological
            analyses, demonstrate the structural, functional and biochemical
            equivalence of the non-plant expressed protein with the plant
            expressed protein.
          □ Acute oral toxicity study completed for newly expressed proteins.




                                          8
Checklist 13: Assessment of Possible Allergenicity (Proteins)
  □ Demonstrate the susceptibility of each newly expressed protein to
     pepsin digestion.
  □ Where a host other than the transgenic plant is used to produce
     sufficient quantities of the newly expressed protein for toxicological
     analyses, demonstrate the structural, functional and biochemical
     equivalence of the non-plant expressed protein with the plant
     expressed protein.
  □ For those proteins that originate from a source known to be allergenic,
     or have sequence homology with a known allergen, testing in
     immunological assays is to be performed where sera are available.




                                 9
                                                                       Annex-1

                   LIST OF GUIDELINES RELEVANT FOR
              RESEARCH/SAFETY ASSESSMENT OF GE PLANTS


1. Recombinant DNA Safety Guidelines, 1990
2. Guidelines for research in transgenic plants, 1998
3. Guidelines for the Conduct of Confined Field Trials of Regulated, Genetically
   Engineered (GE) Plants, 2008
4. Standard Operating Procedures (SOPs) for Confined Field Trials of Regulated,
   Genetically Engineered (GE) Plants, 2008
5. Guidelines for the Safety Assessment of Foods Derived from Genetically
   Engineered Plants, 2008
6. Protocols for Food and Feed Safety Assessment of GE crops.




                                         10
                                                                                                                                                                      Annex-2

                                                                Food & Feed                                                   Environmental
                                                                   Safety                                                          Risk
                                                                Assessment                                                     Assessment
Recommendations for staged




                                                                                                                                                                         concurrently with field trials)
                                                                                         concurrently with field trials)
completion of specific information




                                                                                            Non-field studies (run




                                                                                                                                                                            Non-field studies (run
                                              Before first field trial




                                                                                                                           Before first field trial
and data requirements for the safety
assessment of GE plants




                                                                         Field studies




                                                                                                                                                      Field studies
Description of the genetically
engineered plant
Biology of the non-transgenic host plant
Donor organism information
Bioinformatic analysis: potential toxicity
and allergenicity
Acute oral safety limit study
Pepsin digestibility assay
Protein thermal stability
Subchronic feeding study in rodents (if
required)
Livestock feeding study (if required)
Molecular characterization
Inheritance of introduced trait
Stability of introduced trait
Expression of introduced protein(s)
Compositional analysis
Reproductive and survival biology
Impact on non-target organisms: Tier I
testing
Impact on non-target organisms: Tier 2
testing




                                             11

				
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