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.
II.
III.
Studies that should be completed before Biosafety Research Level I (BRL-I)
trials are undertaken
Field studies that should be completed during BRL-I and/or Biosafety
Research Level II (BRL-II) trials
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.
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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.
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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 nontransformed 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 nontransformed 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.
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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 nongenetically 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.
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.
1
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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 nontransformed 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 agroecosystems 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.
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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 nontransformed
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.
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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.
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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.
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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.
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Annex-2
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
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Non-field studies (run
concurrently with field trials)
Field studies
Environmental
Risk
Assessment
Before first field trial
Non-field studies (run
concurrently with field trials)
Before first field trial
Recommendations for staged
completion of specific information
and data requirements for the safety
assessment of GE plants
Field studies
Food & Feed
Safety
Assessment