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Trade Memoranda
T-4-119 - EXPLANATORY NOTES ON THE INFORMATION USEFUL FOR
SAFETY ASSESSMENTS OF MICROORGANISMS IN FERTILIZERS AND SUPPLEMENTS
September 1997
INTRODUCTION
These guidelines provide an explanation of the information useful for safety
assessments of microorganisms in fertilizers and supplements as listed in Trade Memorandum
T-4-118: Guidelines to safety assessments of microorganisms in fertilizers and
supplements regulated under the Fertilizers Act. Specifically, this document includes
information such as:
- definitions of the items listed in Appendix I of T-4-118,
- the usefulness of this information for a safety assessment, and
- guidance on developing data.
If you have any questions or comments, please contact:
Canadian Food Inspection Agency
Fertilizer Section
Plant Production Division
2 Constellation Crescent
Ottawa, Ontario
Canada
K1A 0Y9
Tel: 613 221-7519
Fax: 613 228-4552;
or by email at fertilizer@inspection.gc.ca.
SECTION I: DESCRIPTION OF PRODUCT AND ITS INTENDED USE(S)
1.1 PRODUCT DESCRIPTION
General description
A general description of the formulated product or the material intended for research,
as appropriate, and include information such as: physical state (e.g. granular, liquid,
powder...), colour, appearance, etc.
List of all ingredients and contaminants
This is simply a list of all the ingredients which are found in the final product,
including carriers, adjuvants, preservatives, sticking agents and contaminants. It should
also identify all extraneous materiel likely to occur in the product.
Concentration of each ingredient in the final product
The number of viable cells or propagules of the Genus, and where known, the species of
each active ingredient in the final product, guaranteed on a weight basis (e.g. CFU’s
per gram, spores per gram...). Amounts of non-biological ingredients in general terms
(e.g. 10% growth culture, 30% milled peat...).
Material Safety Data Sheets (MSDS) for product and/or ingredients
An MSDS is a comprehensive technical bulletin containing detailed information on a
substance or product. It is a basic source of information for familiarizing oneself with
product specifications and it may identify points (such as possible contaminants) which
may need to be examined in greater detail. An MSDS should also provide a detailed
explanation of precautions and protective measures. The Workplace Hazardous Materials
Information System (WHMIS) provides criteria for developing an MSDS.
Brief description of purpose of the ingredients in the product
The purpose of each individual ingredient (e.g. a sticking agent), or combinations of
several ingredients (e.g., growth culture consisting of K2HPO4MgSO4•7H2O,
NaCl, mannitol, yeast extract), as appropriate.
Brief outline of manufacturing process
Summary of the steps taken to combine the individual raw ingredients to manufacture the
final, formulated product. The summary should identify the critical process points, and
describe the measures that are in place to ensure that these critical points will not
decrease the quality of the final product. This will serve as background information
relating to product purity, consistency and safety.
If a standard manufacturing process has not been established for the product (e.g. some
products intended for research), please describe how the product intended to be tested was
made (e.g. culture bacteria at 25oC in growth media the evening before the
intended release date, to a concentration of 1 x 109 CFU/ml; added 50 ml of
culture to 500 g of sterilized peat).
A flowchart diagram accompanying the text description is preferred.
1.2 FOR PRODUCTS INTENDED FOR RESEARCH
Testing protocol
The test protocol describes how the study will be conducted, and includes information
such as: the start date and duration of the test; the experimental design (e.g. treatment,
number of replicates, the crops that will be treated...); any unusual cropping practices
(e.g. repeated cultivation, hand weeding...); etc.
Monitoring procedures
It is important to measure the presence and level of the microorganism at the test site
over the course of the test. Therefore, it is useful to describe how, when and where
samples will be taken before, during and/or after the test period.
Termination procedures
The disposal procedures for treated material and unused product at the completion of,
or during, the test, and post-test plot uses.
Precautionary measures implemented with respect to food, worker and environmental
safety
Precautions that will be taken to mitigate exposure of the environment, of those
handling the product, and of the food chain and consumers, to the microorganism. This may
include information such as procedures for packaging and transporting the microorganism to
and from the test site, procedures to reduce dispersal after application, procedures to
reduce public access and bystander exposure, personal protective measures and equipment
for those applying the product, means of disposal, etc.
Contingency measures
Describe contingency plans to be implemented in the event of accidental release during
application, or in the event that adverse environmental (or other) effects are observed
during the course of the research.
1.3 FOR PRODUCTS INTENDED FOR REGISTRATION
How and in what quantities is the product to be sold (e.g. four 2 litre containers per
box, 200 g bags, etc.)?
Target market
Describe the market for which the product is intended (e.g. consumer, commercial
horticulture, large scale agriculture...)
Product label
The product label must conform to the appropriate Fertilizers Regulations. The
product label may be drafted in typewritten form, and should include a statement of
precautionary measures, where applicable.
Shelf life
The length of time the product can be stored without alteration to its biological or
chemical integrity. This includes not only times under ideal conditions, but a
description of the factors affecting shelf life, how one can tell when the product
expires, and whether the expiry creates a particular hazard. Please include data in
support of the shelf life.
SECTION II IDENTIFICATION & CHARACTERIZATION OF MICROORGANISM(S)
2.1 IDENTIFICATION OF NATURALLY OCCURRING OR PARENTAL
MICROORGANISM
Accurate identification of the microorganism(s) is a key component to the safety
assessment since conclusions regarding the environmental and human health impact of a
product would likely be invalid if the microorganism(s) were incorrectly identified.
Taxonomic names
The identification of the microorganism which includes the following (if applicable):
genus, species, biovar, subspecies, strain and/or type, as well as any changes in
nomenclature.
Criteria and data to substantiate identity
The methods used to identify and classify the microorganism, and the data
substantiating the identification. The methods should reflect the best technology
available, where there is greatest consensus, for the particular group of microorganisms.
For pure cultures, the information should describe the relationship of the
microorganism to pathogens, and be sufficient to distinguish it from related
microorganisms. For mixed cultures, provide identification of each microbial active
ingredient as if each were a pure culture.
Common name(s), if any
Alternative names, abbreviations or acronyms, including names used in other countries.
Strain history
The culture's origin (e.g. environmental isolate, clinical isolate, culture
collection), and the history of the strain during its development (e.g. selection
procedures, culture maintenance...). If the microorganism is an environmental isolate,
describe the habitat and location where the microorganism was isolated (e.g. rhizosphere
of wheat in Saskatoon, Saskatchewan). If the microorganism is obtained from a culture
collection, provide the name, address and telephone number of the culture collection, and
the accession number. Reports and documentation on any history of use of the test
microorganism in agricultural or other practices.
2.2 CHARACTERIZATION OF MICROORGANISMS
2.2.1. FOR NATURALLY OCCURRING OR PARENTAL MICROORGANISMS
This section describes the basic biological and ecological characterization of the
product's microorganism(s), or that from which a genetically modified microorganism is
derived. The information submitted with regard to this section should provide the
knowledge base to evaluate the potential human and environmental effects, and the
environmental fate of the microorganism.
A - Environmental Fate
The intent of environmental fate information is to provide an understanding of the
factors that influence the short and long-term presence of the microorganism in the
environment. If factors that control the survival and multiplication of the microorganism
are known, it is easier to predict that organism's survival and multiplication under
conditions of use.
Growth requirements and characteristics
The relevant conditions and physiological properties affecting persistence,
proliferation and growth of the microorganism. These may include temperature, pH,
nutritional dependence, oxygen requirements, energy sources, susceptibility or tolerance
to antimicrobial agents, sensitivity to environmental factors (e.g. sunlight,
desiccation...), and so on.
List the various forms of the microorganism during its life cycle. This information is
important because the various forms may differ in regard to persistence, proliferation or
dispersal. These may include symbiotic forms, viable but non-culturable forms, dormant
stages, spore production, and so on. Describe the significance of such forms in terms of
the environmental fate of the microorganism when used as directed (e.g. persistence,
dispersal...).
A list of the target species for which the microorganism is intended, and any other
non-target species which may be affected by the microorganism.
Mode of action on host species
How the microorganism exerts its effect on the target species, if known.
Geographical distribution and level of occurrence
Other habitats in Canada where the microorganism is found, or may proliferate or
disperse, and indigenous levels of the microorganism in those habitats.
Controlled environmental fate studies
Fate data is most crucial when the effects data demonstrate either a wide host range or
significant adverse effects (e.g., adverse effects on plants and animals, or other
non-target organisms of environmental and/or economic importance). Environmental fate data
may come from well designed microcosm experiments, mesocosm experiments, or it may come
from other long-term, controlled studies in which the fate of the microorganism is tested
or monitored. Environmental fate test data may include but should not be limited to:
- persistence, multiplication and dispersion of the microorganism under different
conditions and in different environments (land, marine, freshwater, etc.), and;
- the ability of the genetic material introduced into a genetically modified microorganism
to be transferred to other organisms or to persist in the environment.
Presence in association with food or feed plants
Indicate any food or feed crops on which the microorganism is found in nature. As well,
please include any information on any controlled food and feed residue studies that may
have been conducted. This pertains to residues of the microorganism in question as well as
to chemical residues that may be associated with the microorganism or product.
If there is a possibility that a potential mammalian toxin be produced by the
microorganism, the product will be subject to the residue requirements for chemical
products. For information regarding food and feed residue studies, refer to Trade
Memoranda T-4-113 and T-4-113 Supplement. This residue data will be used to estimate the
dietary exposure of humans and livestock to the toxin.
B - Human Health and Environmental Effects
The intent of human health and environmental effects information is to identify the
type and severity of human health or environmental hazards (if any) that the
microorganism(s) has the potential to pose.
- Any toxins produced by the microorganism and any metabolites that are related to toxins
produced by other microorganisms. If there are any known genotoxin(s) produced by related
fungi or actinomycetes, then an appropriate, sensitive analytical technique(s) must be
used as part of the characterization of the microorganism to determine whether the
microorganism in question produces such (a) genotoxin(s).
Nature and degree of adverse effects associated with the microorganism(s)
- The nature and degree of adverse effects includes a description of details and severity
of effects such as: infectivity, toxicity, genotoxicity, pathogenicity, irritation and
sensitization or hypersensitivity potential, and virulence to humans, other animals,
plants, or microorganisms. Report, as well, on any predisposing factors related to the
effect, and on any available epidemiological data. Where no reported effects are found,
describe the steps taken to ascertain the lack of reports of adverse effects (e.g. the
databases searched, the time period search, the search strategy and the keywords used).
- Is/are the microorganism(s) dermatophytic? Is/are the microorganisms related to a known
human dermatophyte? If yes, describe the relationship, and its significance in terms of
the use of the product as directed.
Relationship to microorganism(s) which are associated with adverse health effects
Describe the relationship of the microorganism to other microorganisms associated with
any adverse health effects described above, and its significance in terms of the use of
the product as directed.
Controlled health effects studies
The purpose of effects testing is to identify characteristics such as:
- pathogenicity potential of the microorganism;
- infectivity/pattern of clearance of the microorganism;
- potential toxicological effects of the microorganism, and associated by-products; etc.
- infectivity and toxicity
Infectivity and Toxicity Tests
Several types of controlled studies can be conducted to assess the capability of
microorganisms to exert toxicity, and to cause disease (pathogenicity) and tissue injury
by invasion of and multiplication within, human tissues (infectivity). Some of these are
briefly described below:
Acute oral infectivity and toxicity
This test involves a single high dose of the active ingredient (i.e. microorganism)
administered orally by gavage, to each test animal. This study provides information on the
relative toxicity resulting from a single high exposure by the oral route and on the
inherent infectivity of the organism.
Acute pulmonary infectivity and toxicity
This test involves intratracheal instillation (injection into the trachea) of a single
high dose of the active ingredient (i.e. microorganism) (preferred method). Studies
conducted by conventional inhalation methodology may be acceptable. These would have to
demonstrate that sufficient exposure takes place. This testing provides information on the
relative toxicity resulting from a single high dose exposure by the pulmonary route and on
the inherent infectivity of the organism.
Acute infectivity by intravenous administration
This test applies primarily to bacteria and viruses. It involves a single high dose of
the active ingredient, or microorganism, injected intravenously. This study provides
information on the potential for infection from a single high dose exposure to the
organism when the skin is bypassed as a barrier. It is useful as an indication of the
inherent infectivity of an organism.
Acute infectivity by intraperitoneal administration
This test applies only to fungi and protozoa. If, based on the physical characteristics
of the microorganism, testing by the intravenous route is possible, then the intravenous
route should be considered for fungi and protozoa instead of the intraperitoneal route.
This study involves a single high dose of the active ingredient (i.e. microorganism),
injected intraperitoneally (into the peritoneum i.e. abdominal cavity - preferred method).
The study provides information on the potential for infection from a single high dose
exposure to the organism when the skin is bypassed as a barrier. It is useful as an
indication of the inherent infectivity of an organism.
Acute dermal toxicity
This type of study uses a single high dose of the formulated product applied to
approximately 10% of the body surface area of each test animal for a 24-hour exposure
period. This study provides information on the relative toxicity resulting from a single
dermal application of the material.
Dermal irritation study
Microorganisms and their metabolic by-products, contaminants and formulating agents may
all be capable of irritating the skin. This potential can be assessed in dermal irritancy
testing. The protocol for dermal irritation studies in OECD guidelines is considered
adequate.
Dermal sensitization
Data regarding any type of hypersensitivity reactions that are known or thought to have
occurred during production, testing and manufacturing are to be reported. Please include
the following details: description of the microorganism and formulation; frequency,
duration and routes of exposure to the material; clinical observations (including the type
of reaction noted) and other relevant information.
Genotoxic potential
Fungi sometimes produce toxin(s) and/or metabolic by-product(s) which have genotoxic
potential. If the characterization data indicate a potential for the production of known
genotoxin(s), appropriate and sensitive analytical studies are useful to determine the
presence or absence of such genotoxin(s).
Tissue culture (viral agents only)
This test provides information on the ability of a viral agent to infect (overt,
persistent, latent or abortive), replicate in, transform or cause toxicity in mammalian
cell lines. The test results should report on all of these endpoints.
- Substance to be tested: The most infectious form of the virus should be
used. This is the form that gives optimal infection in the susceptible cell culture or
host organism. The virus should be titred by the most sensitive method available, and in
the most susceptible host system (tissue culture or host organism).
- Cell Lines: For testing purposes, the following cell lines are
recommended: one human cell line (such as WI38); one primary cell type (such as foreskin);
one primate continuous line (such as monkey CV-1). For cell transformation assay, primary
Syrian hamster embryo (SHE)/SA7 system is recommended. One additional cell line should be
utilized to evaluate potential concerns intrinsic to the microorganism and its intended
use. The source and genetic stability of each cell line used should be described.
- Toxicity: Each cell line should be tested and results reported for
efficiency of plating (clonal survival) as a measure of toxicity.
- Transformation: The oncogenic potential of the viral agent is
evaluated utilizing the SHE/SA7 assay system. Appropriate positive and negative controls
must be used. Inoculated cell cultures should be observed for 21 days. If it is
conclusively demonstrated in the infectivity testing that the viral nucleic acid is not
persistent in any of the cell lines tested, this assay may not be useful.
- Infectivity: Each cell line should be exposed to high
multiplicities of the most infectious forms of the virus. The cell cultures should be
observed for 21 days post-inoculation for cytopathic effects. The cells should be assayed
for virus titre and for viral antigen and nucleic acid on days 1, 2, 5, 7, 14 and 21. The
cell culture fluids should be assayed for infectious virus using an appropriate
susceptible host. Appropriate controls should be used (such as inactivated test virus as a
negative control and permissive cell line or host organism as a positive control).
- The enzyme linked immunosorbent assay (ELISA); dot-immunobinding assays; protein blot
immunoassay (Western transfer) or other similar sensitive assays are recommended for
protein determination.
- The dot blot hybridization; in situ hybridization, Southern hybridization and
other sensitive assays are recommended for nucleic acid determination.
Note: The sensitivity and limitations of each assay should be
presented. Any cytopathic effects and/or viral replication observed in tissue culture
should be described.
Controlled environmental effects studies
Environmental toxicology testing is used to determine possible infectivity, toxicity,
pathogenicity, etc. of a microorganism to non-target organisms (e.g. plants, animals,
invertebrates, other microorganisms, aquatic organisms....).
Note: Some of the information describing potential
human/mammalian health effects (see above) may also be important with respect to
environmental safety and may deserve re-iteration or referencing in this section.
Single-species effects testing
Single-species testing may be appropriate where:
- a particular non-target species is of economic importance, an endangered species or
deemed to be of importance, and is in the area of intended use;
- the microorganism is related to one which is known to cause adverse plant or animal
health effects (in this case, the selection of the appropriate organism to be tested will
be dictated by the susceptible host of the related microorganism);
- it is suspected that the microorganism may have an effect on the target organism.
Ecosystem-level effects testing
This type of testing is used to determine the potential impact not on individual
organisms per se, but on the ecosystem itself. This is particularly useful when
effects on individual organisms cannot be measured, or when it is appropriate to look at
the "bigger picture" that includes interactions between individual organisms,
between different types of organisms, and between trophic levels. Data may be obtained
from microcosm/mesocosm studies or field tests designed to measure:
- changes to ecosystem structure (e.g. changes to the relative abundance of various
organisms or categories of organisms, for example, denitrifying bacteria);
- impacts on ecosystem function (e.g. on cycling of soil nutrients or primary production).
2.2.2. FOR MICROORGANISMS MODIFIED BY TRADITIONAL METHODS1
Modification method
The technique(s) used to modify the microorganism (e.g. mutagenesis, conjugation,
transformation, transduction...)
Trait(s) selected
The new trait(s) or characteristic(s) expressed by the genetically modified
microorganism.
Mode of action of the trait (if known)
How the expressed trait(s) or characteristic(s) affect(s) the target organism.
1- Modification to a parental microorganism must be
described. For genetically modified microorganisms, the recipient microorganism must be
identified and characterized as described in Sections 2.1 and 2.2.1. The necessary level
of detail in describing the genetic change will depend on the method of modification. In
cases where specific changes to the microorganism's genome cannot be (reasonably)
determined, a description of the trait(s) and its mode(s) of action is required. However,
in cases where a specific change to the microorganism's genome is known, the source and
function of the genetic material, and the method used to modify the recipient
microorganism should be described
2.2.3. FOR MICROORGANISMS MODIFIED BY MOLECULAR BIOLOGICAL TECHNIQUES
A - Source and function of introduced genetic material
Taxonomic name(s) of donor microorganism(s)
The identification of the microorganism from which the introduced genetic material was
obtained. Please include the following (if applicable): genus, species, biovar,
subspecies, strain and/or type.
Gene transfer potential
Genetic element(s) aiding gene transfer that are present on introduced genetic material
should be described. For genetic elements associated with gene transfer abilities, the
origin of the elements (i.e. the organism from which the DNA was first isolated, according
to the literature) should be included. The ability of the recipient microorganism to
transfer genetic material to other organisms (e.g. via transformation, conjugation,
transduction, transfection...), and the presence of any genetic element(s) which aid gene
transfer (e.g. transposable elements, insertion sequences, mobilizable plasmids,
susceptibility to phages...) should also be described.
Genetic material/gene(s) and gene product(s) inserted in recipient microorganism
The name of the gene, the trait or characteristic encoded by the gene, and the number
of copies of the inserted gene.
Promoter(s) including source(s)
The promoter controlling the expression of the inserted gene, and the original source
of the promoter. If the genetic engineering involves gene rearrangement and the gene is
not inactivated, the original source of the promoter may be the recipient microorganism.
Terminator sequence(s) including source(s)
The sequence terminating the transcription of the inserted gene, and the original
source of the terminator. If the genetic engineering involves gene rearrangement and the
gene is not inactivated, the original source of the terminator may be the recipient
microorganism.
Additional genetic sequences
Genetic material inserted in the recipient microorganism other than the genetic
material coding for the intended function (e.g., marker genes, non-coding open reading
frames, unidentified DNA sequences...).
Resistance to clinically or veterinarily important antibiotics, and potential
impacts
Identify any antibiotic resistance that is conferred to the parental microorganism
through the introduction of genetic material.
B - Strain construction
Flow diagram detailing strain construction
A diagram detailing the names of the donor, recipient and vector DNA, and the steps,
linked in the order of their development, used to construct the final, modified
microorganism (see Appendix I).
Manipulations used to construct the modified microorganism
A description of the manipulations used to construct the final, modified microorganism.
The level of detail provided should be sufficient to allow someone knowledgeable in the
field to repeat the construction. The description should reflect the steps summarized in
the Flow diagram detailing strain construction (above) and include descriptive
information such as:
- a more complete characterization of the vector(s) involved in the manipulations,
including name, type (cloning, expression, shuttle...), and source of vector or vector
components, mobilizability/incompatibility groups, and gene expression in the vector
- methods used to isolate and identify the genetic material introduced into the recipient
microorganism (standard cloning procedures may be referenced rather than described)
- the chromosomal or extrachromosomal location of the gene of interest, or to which
genetic material was deleted or rearranged (e.g. through site-directed mutagenesis)
- methods and results to demonstrate the success of insertion, deletion and/or
rearrangement (e.g. nucleotide sequence adjacent to region of insertion, relevant sequence
data before and after deletion or rearrangement)
- procedures for selection of intermediate constructs
Summary diagram of final genetic construct
A diagram summarizing key features of the final genetic construct (see Appendix I).
Legend for summary diagram of final genetic construct
A brief description, in legend form, indicating all aspects of the details in the
summary diagram of the final genetic construct.
2.2.4. FOR ALL MODIFIED MICROORGANISMS
The expression of the introduced genetic material must be described to more fully
reflect the potential effects of the modification on the human and environmental safety
characteristics of the recipient microorganism.
Alteration(s) to characteristics of parental microorganism
Differences between the characteristics of the parental microorganism and the
genetically modified microorganism attributable to the modification of the parental
microorganism, are to be presented. Particular emphasis should be placed on describing the
possible impact of gene expression on the environment or human health (via food or
user/bystander exposure). Such impacts could consider, for instance:
- traits that might give the modified microorganism an ecological advantage or
disadvantage (e.g. growth requirements, factors affecting host range, persistence,
proliferation, growth or dispersal...)
- infectivity, toxicity, genotoxicity, pathogenicity, virulence, irritation/sensitization,
dermatophytic potential, etc. of the modified microorganism
- the potential health and environmental impact of marker genes conferring resistance to
antibiotics and/or heavy elements (if resistance to clinically or veterinarily important
antibiotics are used, please provide a rationale for their use).
Expression pattern(s) of introduced gene(s)
How the introduced genetic material is expressed in the genetically modified
microorganism (e.g., inducible vs constitutive? If inducible, is the mechanism chemical or
developmental?). Discuss the significance of the expression with respect to the use
pattern of the product and the impact on the environment.
Unintended gene expression or gene suppression in the modified microorganism
Assessment of potential for unexpected gene expression or suppression that may occur in
the recipient microorganism as a result of the genetic modification (e.g. potential
effects of unidentified DNA sequences or non-functional open reading frames...).
Genetic stability
Assessment of potential for reversion to the parental phenotype and genotype (e.g.
reversion and/or mutation rates, rate of extrachromosomal or chromosomal DNA loss, rate of
gene transfer...).
Fate of gene product(s) encoded by introduced gene(s)
Assessment of where the product encoded by introduced gene(s) may end up (e.g.
intact/breakdown/degraded product in the environment, food chains of humans and native
fauna populations...).
2.3 DETECTION AND QUANTIFICATION OF NATURALLY OCCURRING OR MODIFIED
MICROORGANISM
Tests to detect and quantify microorganism
Method(s) by which the microorganism can be detected and quantified from product or
environmental samples. This method should include the sensitivity, specificity,
reliability, practicality and limitations of the detection system, and must allow
third-party identification and quantification of the microorganism(s). Where the
microorganism has been genetically modified, the detection method should be able to select
for the modified microorganism in the presence of the unmodified parental microorganism.
All studies should follow the principles of Good Laboratory Practice (GLP). The GLP
principles of the U.S. EPA (Volume 48, No. 230, 29 November 1983) and OECD Principles of
Good Laboratory Practice (Annex 2 of the Council Decision C(81)30 final) can serve as
guidelines.
APPENDIX I
FLOW DIAGRAM AND SUMMARY DIAGRAM FOR CONSTRUCT ANALYSIS
This appendix provides guidance on how to prepare a flow diagram and a summary diagram
for the final construct. It outlines the key information needed to fully describe the
final, modified microorganism.
Flow Diagram Detailing Genetic Construction
The final, modified microorganism will likely consist of a chromosomal insertion of a
nucleic acid sequence, but may consist of other modifications such as deletions or
rearrangements to the genome, extrachromosomal recombinant plasmid(s), etc. The purpose of
the flow diagram is to illustrate the sequential steps used to construct the final,
modified microorganism. The flow diagram should indicate such aspects as all plasmids and
vectors used in the construction (including relevant names, restriction endonuclease
sites, marker genes...), and genetic manipulations (e.g. cloning, fragment purification,
attachment of linkers, ligation, conjugation, homologous recombination...). An example is
given in Figure 1.
Summary Diagram of Final Genetic Construct
The purpose of the summary diagram is to illustrate the nature and source of the donor,
recipient and vector DNA that is present in the final, modified microorganism. The summary
diagram should indicate such aspects as the name and size of the final, genetic construct,
the coding genes of primary interest including sources, and should show the direction of
transcription, relevant genetic sequences (e.g. promoters, terminators, regulators and so
on, including sources), additional open reading frames including sources, relevant
restriction endonuclease sites, and marker or other relevant nucleic acid sequences (e.g.
those that serve plasmid replication, maintenance and transfer functions). It should
include a summary legend. An example of a final genetic construct is given in Figure
2. Extrachromosomal final construct diagrams would contain the same level of detail
and include information on sequences flanking the site of chromosomal insertion.
This replaces trade memorandum T-4-119 dated October 1996.(Administrative
changes made September 1997 to reflect the changeover to the Canadian Food Inspection
Agency, April 1 1997.)
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