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-4-108 - Efficacy data requirement for 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
Plant Health and Biosecurity Directorate
Crop Inputs Division
Fertilizer Section
59 Camelot Drive
Ottawa, Ontario
K1A 0Y9
Canada
Telephone: 613-773-7189
Facsimile: 613-773-7163

---

Section I: Description of Product and it's 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 K₂HPO₄ MgSO₄.7H₂O, 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

Test site information

Test site information includes two components: identification and characterization.

• Identification: Exact geographical location (i.e. legal land description) or complete postal address, as appropriate, and size of test (e.g. x metres by y metres).
• Characterization: Description of site characteristics (e.g. soil type, nutrient levels, history of previous use, distance to surface or ground water), comparison between natural habitat of the microorganism and the test site, and description of potential growth kinetics of the microorganism at the test site. The history of the site (e.g. organisms previously tested on the site, growth of specific crop to purposely reduce soil nutrient level, such as the growth of corn to reduce soil nitrogen levels) should also be described.

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

Product name

The name of the product as it appears on the product label.

Unit amount/package size

How and in what quantities is the product to be sold (e.g. four 2 litre containers per box, 200 g bags, etc.)?

Use patterns

This information describes how, when and where the product will be used, by specifying:

• all plant species and soil types (if applicable) for which the product is intended
• whether the product will be used on a food or feed crop
• whether the product will be used indoors or outdoors, contained or uncontained, etc.
• mixing information if used in conjunction with other products
• suggested rate(s) of application
• times, frequency and methods of application
• distribution, storage and handling information
• recommended emergency measures
• strategies for re-use, resale or disposal of unused product

Information related to user exposure

This information is related to use patterns but focuses more specifically on exposure of users to the product and its ingredients.

• the amount of product handled by users
• the frequency and duration of handling products
• whether the product is diluted before use
• recommended safety measures directed at those handling/using the product
• possible routes of user/bystander exposure (e.g. skin, inhalation)
• any studies demonstrating exposure of users to the product or any of its ingredients

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 and 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.

Life cycle

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).

Host range

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 - Guidelines to Safety Assessments of Fertilizers and Supplements and to Information to be Submitted in Demonstrating Product Safety and T-4-113 Supplement 1 - Data Requirements for Product Safety Evaluations: Explanatory Notes. 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.

Toxin production

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).

Dermatophytic potential

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.

Potential environmental effects

This pertains to the potential impact on the environment from the intended use of the microorganism. Information may include but should not be limited to:

• enhanced or decreased growth of target and non-target plants
• adverse effects on the health of target and non-target plants and animals
• establishment of competitive but poorly performing microbial inoculants
• host specificity
• displacement of microbial community structure or function
• effects on natural processes (e.g. mineral cycling)
• increased resistance to adverse environmental conditions (e.g. salt or cold tolerance)
• increased resistance to selectable traits (e.g. antibiotic resistance, heavy metal resistance)
• effects of deliberately introduced traits on any of the above.
• effect on biological diversity (e.g. distance to populated or protected areas, identification of, and impact on, endangered or threatened species, or any biological resources of social or economic importance in area of intended use)

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).

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. Extrachromosomal final construct diagrams would contain the same level of detail and include information on sequences flanking the site of chromosomal insertion.