The Thai Draft Guidelines on Contained Uses of GMOs

Table of Contents:CHAPTER 1. SCOPE OF GUIDELINES CHAPTER 2. CATEGORIES OF SMALL-SCALE WORK INVOLVING GENETIC MANIPULATION CHAPTER 3. ROLES AND RESPONSIBILITIES CHAPTER 4. IMPORTATION AND TRANSPORTATION APPENDIX 1. RELEVANT DOCUMENTS APPENDIX 2. ORGANISMS KNOWN TO EXCHANGE DNA BY KNOWN PHYSIOLOGICAL PROCESSES APPENDIX 3. LIST OF APPROVED HOST-VECTOR SYSTEMS APPENDIX 4. INSTRUCTIONS FOR COMPLETING PROPOSAL FORMS APPENDIX 5. TOXINS APPENDIX 6. REQUIREMENTS FOR WORK WITH LIVE VIRAL VECTORS APPENDIX 7. PROCEDURES FOR WORK WITH HAZARDOUS FRAGMENTS OF DNA APPENDIX 8. SUPPLEMENTARY INFORMATION REQUIRED FOR WORK WITH WHOLE PLANTS APPENDIX 9. REQUIREMENTS FOR PHYSICAL CONTAINMENT LEVEL C1 APPENDIX 10. REQUIREMENTS FOR PHYSICAL CONTAINMENT LEVEL C2 APPENDIX 11. REQUIREMENTS FOR PHYSICAL CONTAINMENT LEVEL C3 APPENDIX 12. REQUIREMENTS FOR PLANT-HOUSE LEVEL PH1 APPENDIX 13. NOTE ON PLANT HOUSES WITH HIGHER CONTAINMENT THAN PH1 APPENDIX 14. EQUIREMENTS FOR PLANT-HOUSE LEVEL PH3 APPENDIX 15. REQUIREMENTS FOR EXPERIMENTS INVOLVING TRANSGENIC ANIMALS APPENDIX 16. REQUIREMENTS FOR HOUSING OF INFECTIOUS TRANSGENIC ANIMALS APPENDIX 17. DESIGN OF TRANSGENIC-ANIMAL FACILITIES APPENDIX 18. REQUIREMENTS FOR ANIMAL-CONTAINMENT LEVEL C1A APPENDIX 19. REQUIREMENTS FOR ANIMAL-CONTAINMENT LEVEL C2A APPENDIX 20. NOTE ON TRANSPORT

CHAPTER 1 SCOPE OF GUIDELINES

1. These Guidelines apply to any experiment carried out in laboratories of government, state enterprises, private organizations, or companies and involving the construction and/or propagation of viroids, viruses, cells or organisms of novel genotypes produced

by genetic manipulation which are either unlikely to occur in nature or likely to pose a hazard to public health or to the environment. 2. If an investigator is uncertain if the work he/she is proposing to do falls within the scope of these Guidelines, a description of the proposed work should be submitted to the Institutional Biosafety Committee (IBC) for written clarification before work commences. If there is no IBC in the firm or institution, a description of the work proposed should be sent to the National Biosafety Committee (NBC).

CHAPTER 2 CATEGORIES OF SMALL-SCALE WORK INVOLVING GENETIC MANIPULATION

Small-scale work in the field of genetic manipulation and biotechnology is classified according to risk assessment.

Category 1 - Work which is exempted.

Category 2 - Work which may involve a low level of hazard to researchers, the community and the environment.

Category 3 - Work which may involve a high level of hazard to researchers, the community, the environment or, in the case of human gene therapy, to the patient. Included in this category is work for which the nature or the degree of hazard may be uncertain.

2.1. Category 1

Work which needs no approval from the National Biosafety Committee.

2.1.1. The following types of experiments are classified as Category 1 work:

A Any experiment involving the fusion of mammalian cells which does not generate a viable organism, for example, the creation of hybridomas to produce monoclonal antibodies (Appendix 1). B Protoplast fusion between non-pathogenic microorganisms. C Protoplast fusion or embryo rescue involving plant cells. D Work with self-exchanging organisms in which the donor species is also the host species or a species known to exchange genetic information with the host species under natural conditions (Appendix 2). E Any experiments involving approved host-vector systems (Appendix 2) if the donor DNA:

is not derived from microorganisms able to cause disease in humans, animals or plants;

does not code for any protein which regulates the growth of mammalian cells (for example, the product of an oncogene), a cytotoxic protein or a toxin for vertebrates with an LD50 of less than 100 µg/kg;

does not comprise or represent more than two-thirds of the genome of a

virus and is not being used in an experiment in which the genetic material missing from the viral genome, and essential for producing infection, is available in the cell into which the incomplete genome is introduced or is made available by subsequent breeding processes.

Exempted work should be carried out under conditions of standard microbiological laboratory practice. If pathogenic organisms are used, the containment level appropriate to the pathogen should be employed, and personnel should have appropriate training.

2.1.2. IBC Notification

A Scientists who believe that their work falls into any of the above Category 1 exemptions are nevertheless required to notify their Institutional Biosafety Committee (IBC) of the proposed project. If the institution does not have an IBC, scientists should notify the NBC by submitting a description of the proposed work before commencement. B Forms for IBC notification for exempted work may be devised by IBCs. A sample of such a form is shown in Appendix 4. C Any substantive change in experiments already granted a "Special Exemption" which may affect the exemption status requires a new proposal form to be submitted to the IBC for notification. If the IBC endorses the change in the proposed work, it will send it on to the NBC.

2.2. Category 2

Experiments which involve a low level of hazard to laboratory personnel, the community or the environment.

Such work requires at least C1 or, in the case of work with whole plants or animals, PH1 glasshouse or C1 animal physical containment. Some work may require additional precautions or a higher level of containment because the donor DNA or its components are by their nature hazardous or infectious. Other work may require special containment features; the housing of transgenic animals is an example. Procedures for C1 and other containment levels are in Appendices 9-19.

The IBC and the investigator should identify all potential sources of hazard and their nature and determine any additional procedures and conditions appropriate for work in this category.

The following subcategories of work must be described in proposals submitted to the IBC for determination of the appropriate working and containment conditions, and may begin only after IBC assessment and approval. The IBC should subsequently forward the proposal and assessment forms to the NBC for information.

The following types of experiments are classified as Category 2 work:

A Genetic manipulation work involving whole animals (including non-vertebrates, but excluding microorganisms). Genetic manipulation of the genome of the oocyte or zygote or early embryo by any means to produce a novel whole organism (Appendix 15). B Genetic manipulation work involving the production of modified whole plants. Note that the investigator needs to submit a supplementary information form for work involving whole plants (Appendix 8). C Work with non-approved host-vector systems, i.e., other than those listed in Appendix 3. Work with approved host-vector systems, where the gene inserted:

is a pathogenic determinant; is uncharacterized DNA from microorganisms able to cause disease in

humans, animals or plants; encodes a protein involved in regulating cellular growth (for example, an

oncogene).

2.3. Category 3

Work which may involve a hazard to researchers, the community, the environment or, in the case of human gene therapy, to the patient, or work for which the nature or the degree of hazard may be uncertain.

The level of physical containment required will vary according to the nature of the work and its assessed hazard. In some cases, C1 may be considered sufficient, while in others higher containment levels may be necessary. This category of work requires assessment by the NBC which must then advise the IBC.

The following types of experiments fall into Category 3:

A Work with toxin producers: Work with DNA encoding a toxin for vertebrates having a LD50 of less than 100 µg/kg. (Appendix 5). Work involving high-level expression of toxin genes even if the LD50 is greater than 100 µg/kg. Work with uncharacterized DNA which may include toxin sequences from toxin-producing organisms. B Experiments involving viral vectors whose host range includes human cells and which contain one or more inserted DNA sequences coding for a product known to play a role in the regulation of cellular growth or to be toxic to human cells (special conditions for working with viral vectors encoding oncogenes are contained in Appendix 6). C Experiments using, as host or vector, microorganisms which are able to cause disease in plants, humans or animals, except for those listed as approved hosts or vectors (Appendix 3). Included in this subcategory are experiments using defective vector-host virus combinations which have the potential to regenerate a non-defective recombinant virus. D Introduction of genes determining pathogenicity in microorganisms other than the approved hosts listed in Appendix 3. E Cloning or transfer of complete viral genomes, viroids or of fragments of a

genome capable of giving rise to infectious particles pathogenic to humans, animals or plants. Generally, work involving less than two-thirds of the complete viral genome, or work with a genome lacking gene activity for a vital component of replication or packaging not supplied by the experimental system would fall outside this subcategory. F Experiments involving recombinations between whole viral genomes, viroids and/or complementary fragments of such genomes where one or more fragments contain virulence or pathogenic determinants. This subcategory includes experiments which alter host ranges of pathogens or which may increase their virulence or infectivity. G All proposals for human gene therapy work. H Any experiment to inject a fragment or the whole genome of a virus into an embryo to produce a transgenic animal secreting or producing infectious viral particles (Appendix 15). I Experiments not falling into Category 1, 2 or 3 but which fall under the scope of the Guidelines as defined in Section 1.

CHAPTER 3 ROLES AND RESPONSIBILITIES

Roles and responsibilities of various regulatory authorities.

In order to oversee and review the safety aspects of genetic manipulation work and biotechnology, three levels of regulatory authorities with specific roles and responsibilities are established:

the National Biosafety Committee (NBC) Institutional Biosafety Committees (IBCs) Principal Investigators

3.1. The National Biosafety Committee

The National Biosafety Committee was established by the Minister of Science, Technology and the Environment on 22 January 1993 according to the recommendations of the National Committee for Genetic Engineering and Biotechnology, which has established guidelines for regulation and/or advice in biotechnology and genetic engineering work so that potential risks and hazards to the community and the environment can be prevented.

3.1.1. Roles of the National Biosafety Committee

A Maintain an overview of the biosafety factors associated with innovative genetic manipulation techniques. B Identify and keep under review classes of work which have an undefined risk level. C Alert regulatory authorities to the existence of risk factors.

D Provide specialist technical advice on specific biosafety matters to organizations using these techniques and to regulatory agencies. E Prepare or, as appropriate, assist with the preparation of codes, standards or guidelines for the assessment and management of biosafety risk factors, whether for the Committee's own overseeing activities or to assist regulatory agencies. F Participate in public discussions on the biosafety of these techniques. G Liaise with agencies overseas to ensure that, as far as practicable, Thai guidelines and regulations are in harmony with international practice.

3.1.2. Responsibilities of the National Biosafety Committee

With regard to the administration of these Guidelines, NBC will:

A Provide advice to Institutional Biosafety Committees (IBCs) on Category 3 proposals (Chapter 2). B Provide advice on any other category of proposal if such advice is sought by IBCs. C Inspect and certify C2 and C3 laboratories and facilities, facilities with C1-LS (large scale) and C3-LS containment levels and plant and animal facilities with equivalent levels. D Provide signs for facilities, proposal forms, IBC assessment forms, copies of Guidelines to IBCs. E Refer matters concerning the safety of genetic manipulation to relevant government agencies, institutions and firms, where appropriate. F Protect information which may have a commercial significance. If investigators wish to restrict access to such information, they must mark each of the relevant pages of the proposal "Confidential".

3.1.3. Certification of C2, C3 and PH2 Laboratories

C2 and C3 laboratories (including animal containment facilities with these levels) and PH2 facilities are certified by the NBC. Following receipt of the report, the NBC will ether certify the laboratory or advise the IBC of any problems preventing certification. After certification of a C2, C3 or PH2 laboratory, the IBC must undertake regular inspections as required for C1 laboratories. In addition, the NBC reserves the right to inspect laboratories and facilities at any time without notice.

3.1.4. Secretariat Contact

The National Biosafety Committee's postal address is:

National Centre for Genetic Engineering and Biotechnology,Ministry of Science, Technology and the Environment,Building Rama VI, Bangkok 10400, THAILAND. Telephone: (066)-2-2452498-9. Facsimile: (066)-2-2464850.

3.2. Responsibilities of Institutions

Any institution, firm or organization which conducts genetic manipulation work, undertakes importation of organisms arising from such work or produces such organisms or plans to release such organisms into the environment or for sale is required to set up an Institutional Biosafety Committee (IBC), provide such resources and facilities as are necessary for safe work in laboratories and for the IBC to undertake its duties adequately and ensure by recruitment procedures and other measures that adequate supervision of work takes place. Institutions may consider making compliance with these Guidelines a term of employment contracts, where appropriate. Institutions conducting large-scale or industrial-scale work should have a Biological Safety Officer (BSO).

The National Biosafety Committee recognizes the difficulties that small institutions and small firms have in setting up an IBC. These firms may choose the option of being supervised by the nearest IBC or by an IBC in the same province. Such arrangements need to be formalized between the institutions concerned and notified to NBC. A representative member of the smaller institution needs to liaise closely with, or be a member of, the IBC.

The responsibilities of institutions are:

To review the qualifications and experience of personnel involved in research proposals, to ensure that they are adequate for good microbiological practice and for the supervision of junior personnel.

To maintain a register of approved projects on genetic engineering and their assessment and projects exempted under section 2.1 of these Guidelines.

To maintain a list of people working in laboratories requiring levels of containment and ensure that new personnel are familiar with procedures at the relevant containment level and the correct use of laboratory equipment.

At the time of establishment, the IBC is to provide the NBC with a completed "Institutional Biosafety Committee Information" form. This form is available from the National Biosafety Committee Secretariat.

The IBC report must include the following information:

Chairman and Secretary; Biological Safety Officer, where applicable; Membership of Committee; List of current Category 1, 2 and "Special Exemption" proposals; List of certified C2, C3 and C1-LS and C3-LS laboratories; List of certified plant houses and transgenic-animal facilities; Any significant accident or incident relating to health of workers which may

reasonably be attributed directly to genetic manipulation work;

Any other matter which the IBC deems necessary to draw NBC's attention to.

To fulfil these reporting requirements, the NBC will send the IBC its report for amendment.

Many institutions carrying out microbiological research take, as a matter of routine, serum samples from workers at regular intervals. The samples are stored and are available for later diagnostic tests on workers exposed to accidents or who develop unexplained illnesses. If this practice is not already followed, it is recommended that it be instituted, particularly for workers involved in projects requiring C3 physical containment facilities.

The IBC should record accidents and the action taken in dealing with accidents. If the IBC Chairman is satisfied that an accident or incident occurred which was directly attributable to work with genetic manipulation, and was of sufficient significance, he/she should make a report to the NBC and the head of the institution. An example of such an incident might be the deliberate failure to comply with these Guidelines, or any incident which may have resulted in a risk to human health or to the environment.

3.2.1. Recruitment and Training of Staff

An institution, firm or its recruitment section should ensure that staff recruited to work in laboratories are informed of hazards, have adequate training to ensure that their work is carried out under these Guidelines and have access to the IBC Chairman or Biological Safety Officer for advice.

The IBCs are vital components of the overall monitoring and surveillance of genetic manipulation work, and of the administration of these Guidelines. The calibre and experience of members on the IBC should be such that it can competently carry out its duties. The Chairman of the Committee should be of sufficient standing in the institution for decisions and advice by IBC to be effective.

3.2.2. Composition

The IBC should include:

five specialists with the requisite knowledge and expertise to assess, evaluate and oversee work being carried out in the institution;

the Biological Safety Officer, where applicable; an engineer with expertise in testing biological safety facilities and equipment; at least one informed or interested external member from the wider community

who need not have a technical background.

3.2.3. Roles of IBCs

The main functions of IBCs are to: A Undertake the assessment and review of all the research proposals it receives, including changes to "Special Exemption" projects, in order to identify all potential hazards to the researchers, the community and the environment and to provide advice to the investigators on these hazards and their management. B Determine containment and procedures for all experimental work falling into NBC Categories 1 and 2 (Chapter 2) and for the housing, storage and transportation of genetically manipulated organisms falling under these Guidelines. C Send legible original typewritten proposal forms and IBC assessment forms to the NBC for review and assessment of experiments falling into Category 1 and 3 and ensure that NBC advice is complied with. Send original typewritten proposal forms and assessment forms to NBC for notification of experiments falling into Category 2 (Appendix 4) for instructions on filling in proposal and IBC assessment forms. D Inspect and certify C1 laboratories, transgenic-animal facilities, infectious- animal holding facilities and PH1 glasshouses before they are used for genetic manipulation work. Certify laboratories and facilities of higher containment levels than C1 (also, the IBC needs to conduct inspections and monitor procedures in all the institution's laboratories and animal and plant facilities requiring any level of containment). At least annual inspections of these facilities should be undertaken to ensure that they continue to meet the relevant containment standards and requirements. Detailed requirements for these facilities are given in Appendices 9-19. Signs are available from the Secretariat. E Monitor the ongoing work and give periodic advice to the investigators when required. F Take responsibility for drafting rules and make decisions on specific procedural safety matters in the institution.

3.2.4. Biological Safety Officer

It is recommended that institutions either appoint a Biological Safety Officer (BSO) or assign such duties to the IBC. If institutions have more than one officer, for the purposes of these Guidelines, only one name from each institution is to be submitted to NBC in the required annual reports. The officer, ideally, should have experience of work performed in containment conditions. The officer should be adequately trained and be able to offer advice on, or participate in, training of new staff of laboratory personnel. Appropriate deputizing arrangements should be made when the officer is on leave.

The Biological Safety Officer or the IBC Chairman should act as adviser to the head of the institution or firm in all matters relating to containment, biological hazards and the safety of staff. Regular safety audits and the supervision of a regular testing program for appropriate pieces of equipment should be undertaken by the BSO or the IBC.

Conflicts of Interest

The composition of IBCs is such that they often include members with specialist expertise who themselves originate proposals. It is undesirable for members to assess their own proposals, and these Guidelines recommend that the IBCs have sufficient scientific members so that they are not dependent on the advice of the person submitting the proposal. However, to ensure that no conflicts of interest arise, it is recommended that proposals be assessed in the absence of the originator.

It is also becoming more usual for investigators to have, in addition to their usual responsibilities, other commercial affiliations or interests. It is recommended that in such cases members should declare their interests and leave the room when that particular item of the agenda is being considered.

3.2.5. Monitoring of Work

The IBC is to ensure that NBC's and its own advice on specific proposals is conveyed to the Principal Investigator(s) and, where necessary, is acted upon. The IBC is to visit laboratories and facilities from time to time to monitor safety aspects of ongoing projects.

3.3. Responsibilities of Principal Investigators

The Principal Investigator must be thoroughly familiar with the requirements of these Guidelines and must ensure that, where applicable, they are followed for any project for which he/she is responsible. In particular, the Principal Investigator must:

3.3.1. Assess the proposal to determine if it falls within the Guidelines. If in doubt, the Investigator should consult the IBC or, in the absence of an IBC, should contact in writing the NBC.

3.3.2. Notify the IBC on proposals which the Investigator considers to be Category 1 under Chapter 2.

3.3.3. Provide any information on the proposal and its conduct that the IBC may require for its assessment and monitoring activities.

3.3.4. Carry out NBC and IBC advice and recommendations on proposals.

3.3.5. Send the complete proposal of work to the IBC responsible for the laboratories where the work is to be carried out, before starting any work on any project which falls under these Guidelines, and ensure that work does not start until approval is given by the IBC.

3.3.6. Forward a new proposal form to the IBC before any substantial change is made to the components of the experimental system of a proposal which may affect its categorization or its exempt status.

3.3.7. Carry out work under conditions of physical containment approved by the IBC or as advised by the NBC in the case of Category 3 proposals (see Chapter 2).

3.3.8. Ensure that students, subordinates and other co-workers are aware of the nature of potential hazards of the work and have received appropriate training. Arrange for training, if necessary.

3.3.9. Notify all changes in the project team to the IBC.

3.3.10. Report immediately to the IBC all accidents and unexplained illnesses or absences.

3.3.11. Advise the IBC of any intention to import biological material falling under these Guidelines (see also Chapter 4, Section 4.5).

3.4. Sanctions

Non-compliance with these Guidelines by Investigators may result in withdrawal of grants by funding authorities.

Registration for tax incentives for private sector funding of research and development may also be conditional upon compliance with the Guidelines.

A research institution which continues to breach substantive requirements of the Guidelines or exposes its staff or the community to undue risks will be reported to the Minister, who may make a public statement.

CHAPTER 4 IMPORTATION AND TRANSPORTATION

4.1. Packaging and Transport of Samples

4.1.1. The basic requirement for any mode of transport of viable microorganisms is that they should not be harmful to humans or the environment if the packaging leaked or were damaged.

4.1.2. For international postage, the revised conditions required by the Non-Infectious and Infectious Perishable Biological Substance Services agreed by the International Postal Union must be observed.

4.2. Transport within and between Institutions

Care must be taken regarding the transport of material likely to contain live recombinant or genetically manipulated organisms, for example, transport between laboratories or to autoclaves elsewhere in the building. Any container of viable organisms must be transported within a secondary unbreakable and closed container which can be readily decontaminated.

4.3. Transport of Transgenic Animals

4.3.1. With regard to the transportation arrangements for transgenic animals, two principles must be paramount:

the need to prevent the animals from escaping, especially with regard to reasonable contingencies such as accidents en route, so that they will not interbreed with feral populations;

the need to ensure that they are properly identified and duly arrive at the intended destination, and to ensure that a competent biologist with some experience in handling transgenic animals takes delivery of them.

4.3.2. The IBC may institute whatever procedures or rules it considers appropriate to meet these conditions. It may be necessary for the IBC to inspect the arrangements for transportation to satisfy itself that the above principles are adhered to and that any additional conditions which the IBC considers appropriate are met (Appendix 17).

4.4 Provision of Genetically Manipulated Material to Other Research Workers

4.4.1. Investigators supplying people with genetically manipulated DNA preparations must ensure that recipients new to the genetic manipulation field are made aware of the existence of these Guidelines and of the need to comply with them. When supplying such organisms to scientists overseas, information on containment and any special conditions should be provided to the recipient.

4.4.2. Investigators must supply information on the origin of genetically manipulated materials.

4.5. Importation of Samples

4.5.1. The intention to import a genetically engineered viral genome, microorganism, plant or animal is subject to these Guidelines. Investigators should consult the IBC about their intention to import such samples and submit a proposal for assessment if appropriate.

4.5.2. The import or export of disease-causing organisms must follow the "Diseases and Animal Toxic Substances Acts BC. 2525".

APPENDIX 1 RELEVANT DOCUMENTS

1. Department of Administrative Services, Australia, 1985. Guidelines for Small-Scale Genetic Manipulation Work.

2. Department of Administrative Services, Australia, 1990. Guidelines for Large-Scale Work with Genetically Manipulated Organisms.

3. NIH (National Institutes of Health), USA, 1986. Guidelines for Research Involving Recombinant DNA Molecules.

4. Department of Community Service and Health, Australia, 1988. Guidelines for the Preparation and Presentation of Applications for General Marketing of Monoclonal Antibodies for Use in Humans.

5. Anon., 1988. Laboratory Containment Facilities for Genetic Manipulation Experiments.

6. Anon., 1968. Guidelines for the Categorization of Genetic Manipulation Experiments.

7. OECD, 1986. Recombinant DNA Safety Considerations. OECD Publications Service.

8. Commonwealth of Australia, 1988. Infection-Control Guidelines - Acquired Immune Deficiency Syndrome (AIDS) and Related Conditions.

9. Department of Primary Industry, Australia, 1983. Regulatory Control of Veterinary Drugs. Australian Government Publishing Service.

10. Anon., 1981. Biological Safety Cabinets. Part I: Biological Safety Cabinets (Class I).

11. Anon., 1965. Biological Safety cabinets. Part II: Laminar- Flow Biological Safety Cabinets (Class II) for Personnel and Product Protection.

12. Department of Primary Industries and Energy, Australia, 1985. Requirements for Clearance of Veterinary Chemicals. Australian Government Publishing Service.

13. Department of Primary Industries and Energy, Australia, 1985. Requirements for Clearance of Veterinary Drugs. Australian Government Publishing Service.

14. Health and Safety Executive, 1984. Categorization of Pathogens According to

Hazard and Categories of Containment.

15. US Department of Health and Human Services, 1984. Biosafety Guidelines for Microbiological and Biomedical Laboratories. US Government Printing Office.

16. Collins, C.H., 1986. Laboratory-Acquired Infections.

17. WHO (World Health Organization), 1983. Laboratory Biosafety Manual. WHO Distribution and Sale Service.

18. Anon., 1983. Code of Good Manufacturing Practice for Therapeutic Goods. National Biological Standards Laboratory, Australia.

19. Anon., 1985. Code of Practice for the Care and Use of Animals for Experimental Purposes. National Health and Medical Research Council, Scientific and Industrial Research Organization and Australian Agricultural Council. Australian Government Publishing Service.

20. Department of Health and Community Services, Australia, 1987. The National Health and Medical Research Council Statement on Human Experimentation and Supplementary Notes.

21. National Health and Medical Research Council, Australia, 1987. Ethical Aspects of Research on Human Gene Therapy.

22. Anon., 1986. Laboratory Biosafety Guidelines, AIDS Task Force.

23. Sly, L.I., Postal Regulations: Packing and Shipping of Cultures; Quarantine.

APPENDIX 2 ORGANISMS KNOWN TO EXCHANGE DNA BY KNOWN PHYSIOLOGICAL PROCESSES

Organisms known to exchange DNA by known physiological processes are those within each of the following sublists, and the vector used must not contain DNA inserts from organisms not included in the sublist in question. Investigators must submit the proposals to the National Biosafety Committee if DNA exchange is with other, unlisted organisms.

Sublist A

Alcaligenes (2) Campylobacter jejuni (2)Campylobacter coli (2) Campylobacter fetus (2)Citrobacter (including Levinea)

Enterobacter ErviniaEscherichiaKlebsiella Pseudomonas aeruginosaPseudomonas fluorescens Pseudomonas mendocina (3) Pseudomonas putidaRhizobium (2) Salmonella (including Arizona) Shigella Serratia marcescensYersinia enterocolitica

Sublist B

Bacillus amyloliquefaciensBacillus aterrimus Bacillus globigii Bacillus licheniformisBacillus nato Bacillus niger Bacillus pumilus Bacillus subtilis

SubIist C

Streptomyces aureofaciensStreptomyces coelicolorStreptomyces rimosus

Sublist D

Streptomyces cyaneuw Streptomyces griseus Streptomyces venezuelae

Sublist E

One-way transfer ofStreptococcus mutans orStreptococcus lactis DNA into Streptococcus sanguis

Sublist F

Streptococcus faecalisStreptococcus mutans Streptococcus pneumoniae Streptococcus pyogenes Streptococcus sanguis

Sublist G

Bacillus cereus Bacillus thuringiensis

APPENDIX 3 LIST OF APPROVED HOST-VECTOR SYSTEMS

The objective of biological containment is to minimize the chances of the host and the vector to survive outside the laboratory, and of the vector to be transmitted from the propagation host to a non-laboratory host.

This Appendix lists the host-vector systems currently approved as providing a level of biological containment.

1. Bacteria

HostEscherichia coli K12 or a derivative which does not contain conjugative plasmids or generalized transducing phages.

Vector1. Non-conjugative plasmids.

2. Bacteriophage lambda, lambdoid, Ff (eg M 13).

Host Bacillus subtilis or Bacillus licheniformis. Asporogenic strains with a reversion frequency of less than 10-7.

Vector Indigenous Bacillus plasmids and phages whose host range does not include B. Cereus or B. anthracis.

Host Pseudomonas putida strain KT 2440

Vector

Certified plasmids: pKT 262, pKT 263, pKT 264.

HostStreptomyces specified species: S. coelicolor, S. lividans, S. parvulus, S. griseus.

Vector 1. Certified plasmids SCP2, SLP1,SLP2, PIJ101 and derivatives.

2. Acitnophage phi C3 1 and derivatives.

2. Fungi

HostNeurospora crassa specified strains modified to prevent aerial dispersion.

VectorNo restriction.

HostSaccharomyces cerevisiae.

Vector No restriction.

3. Tissue culture

HostMammalian (including human) cells.

VectorNon-viral vectors or defective viral vectors (including retrovirus or retroviral-helper combinations) that cannot infect human cells. Non-tumorgenic disarmed.

Host Plant-cell culture.

Vector Ti plasmid vectors in Agrobacterium tumefasciens and non-pathogenic viral vectors.

Note:The above approved hosts may also be used in experiments where DNA is inserted into the host cell without the use of a biological vector (for example, by mechanical, electrical or other means) if the DNA is:

not derived from microorganisms able to cause disease in humans, animals or

plants; does not code for any protein which regulates the growth of mammalian cells

(for example, oncogene products), a cytotoxic protein or a toxin for vertebrates with an LD50 of less than 100 µ /kg;

does not comprise or represent more than two-thirds of the genome of a virus and is not being used in an experiment in which the genetic material missing from the viral genome and essential for producing infection is available in the cell into which the incomplete genome is introduced or is made available by subsequent breeding processes.

Such a system, with an approved host and the DNA meeting these conditions, would constitute an approved host-vector system for the purposes of these Guidelines, and hence would fall under Category 1 work.

APPENDIX 4INSTRUCTIONS FOR COMPLETING PROPOSAL FORMS

The Institutional Biosafety Committee will use the information provided in the proposal form to determine the category into which the project fits and the containment level. The National Biosafety Committee will use the information in the form to assess proposals falling into Category 3. In order to enable the NBC and the IBC to carry out these functions, a clear statement of what the applicant proposes to do is needed, and if this cannot easily be fitted into the space provided, a separate description should be attached. The following suggestions are intended to ensure that the NBC has sufficient relevant information to make a prompt decision on the application.

Title of Project and Aims

When describing the aim of the proposal, include a brief description of the main steps involved. If both immediate and long-term, broader aims are included, make clear what component of the work needs immediate IBC approval (or NBC advice).

If the project is complex and likely to take several years to complete, it will help if the work to be undertaken first is described separately. If recombinant DNA is ultimately to be put into whole animals or plants, or into bacteria not listed in Appendix 3 of Guidelines, it may well be that approval can be given in the first instance for cloning and characterization of specific genes or other DNA, with approval for later stages being possible only after such characterization. If the stages are made clear to the Committee, the approval or advice for the first stage may be given to enable the work to start.

If there is an intention to import biological material which falls under the Guidelines, the title may read:

"Intention to import......"

Source of DNA

If already cloned, please give details of the clone: who made it, how it was made and what is known of its properties.

If several genes or species, etc. are to be used, list all of them, because one proposal may cover them all. For example, if appropriate, request for chickens, ducks and other avian species. This will eliminate the need for repeated applications.

Do you seek approval to use DNA, but not approval to grow it? If so, please state the source of your DNA.

Host Organism

If more than one host is to be used, particularly if different containment levels apply, clarify when and how each is to be used. Comments made previously on dividing a project into stages may be relevant.

Vectors

Make the description for prokaryotic vectors as broad as is necessary to cover the intended work. For example, say "non-conjugative plasmids such as pBR322 and pUC9" if the project is likely to require a range of specific vectors during its progress. If only pBR322 and pUC9 are requested, the approval will be limited to the two vectors and will not cover the many closely related vectors which may turn out to be more useful.

The description of vectors should not only comprise a series of letters, symbols and numbers. Some words describing its properties are required.

In the case of retroviral vectors, be specific and say what is known about their properties and provide details of the construct, if appropriate, on a separate sheet of paper, including a genetic map and/or a description.

Details of Personnel

For the "full details" requested, please include the extent and nature of relevant experience on a separate sheet of paper. This paper need not be sent on to the NBC. The personnel involved needs to be checked only by the IBC.

The IBC may obtain the proposal and assessment forms from the Secretariat of the NBC. The address is:

National Biosafety Committee National Centre for Genetic Engineering and Biotechnology

Ministry of Science, Technology and the Environment Building Rama VI Road, Bangkok 10400

Proposal Form for Assessment of Work under Exempt Category (Category 1)

Name (Principal Investigator)

Main experimental procedure (description).

Reasons for the project to be classified as category 1 (description with reference to Category 1 work).

Signature.

Date.

Proposals Form for Assessment of Small-Scale Genetic Manipulation Work

1. Name and business address of principal investigator. 2. Name of co-investigators. 3. Title of the project. 4. Objectives of the project. 5. Details of biological system. 5.1. Describe the biological source of the donor DNA to be used. 5.2. Describe the host organism or tissue to be used. 5.3. Describe the vectors or methods to be used. 5.4. What is the biological containment offered by the host-vector system. 6. Into which Category of work established by the Guidelines does this work fall? 7. Where will this work be conducted? 8. What is the certified containment level of this facility? 9. Do you have approval to use this facility? 10. Proposed date of commencement of work. 11. Details of personnel involved in this project (experience/duties). 12. Signature of Principal Investigator submitting this proposal.

Assessment of a Proposal

Section A: Assessment by IBC

1. Names of Principal Investigator(s). 2. Project title. 3. The following information has been checked and approved: 3.1. The aim. 3.2. Biological system. 3.3. Physical containment facility.

3.4. Details regarding the personnel involved in the project. 4. The training and experience of the project team are considered adequate for carrying out this type of work. 5. IBC assessment of: 5.1. The Category of work (1/2/3). 5.2. Physical containment (C1/C2/C3/etc.).

Section B: Request for NBC Advice

6. The IBC has assessed the attached proposal as described above and is forwarding it to the National Biosafety Committee for advice (Category 3). Specific advice is required regarding: Signature of IBC Chairman. Date.

Section C: IBC Decision

7. This proposal has been assessed as described above and endorsed/approved by the IBC. The following conditions, in addition to those specified in the Guidelines, must be adhered to during the conduct of this work: Signature of IBC Chairman. Date.

APPENDIX 5TOXINS

Work involving the cloning of genes from organisms which synthesize toxins for vertebrates with an LD50 of less than 100 µ g/kg requires approval from the National Biosafety Committee for work to commence.

Examples of toxins with an LD50 of less than 100 µ g/kg:

AbrinBacillus anthracis lethal factor Bordetella pertussis toxin Cholera (see Vibrio cholerae) Clostridium botulinum toxins Clostridium perfringens epsilon toxin Clostridium tetani toxin Corynebacterium diphtheriae toxins Escherichia coli heat-labile (LT) enterotoxin and LT -like toxin Oxygen-labile haemolysins such as streptolysin 0 Pasteurella pestis marine toxins Pseudomonas aeruginosa exotoxin A Ricin Shigella dysenteriae toxin Staphylococcus aureus determinants A, B and F, alpha and beta

toxin, exfoliative toxin Vibrio cholerae (comma) toxin and toxins neutralized by antiserum monospecific for cholera toxin (e.g., heat-labile toxins of E. coli, Klebsiella and other related enterotoxins) Yersinia enterocolitica heat-stable toxin

APPENDIX 6 REQUIREMENTS FOR WORK WITH LIVE VIRAL VECTORS

1. General Considerations

A variety of live viral vector has been developed for efficient transfer of genetic material into cells. Hazards associated with these vectors depend on:

the host range of the virus; its infectivity and availability for repeated rounds of infection; whether its genetic material becomes inserted into the host cell; the nature of the foreign genetic material inserted into the virus.

The following notes address the use of retroviral vectors, but similar considerations will apply to the use of other live viral vectors for genetic manipulation work. Most experiments using live infectious viruses as vectors fall under Category 3 of the Guidelines and require assessment by the National Biosafety Committee before work commences.

2. Retroviral Vectors

Retroviral vectors have been developed for the efficient introduction of genes into various animal cells. In principle, they are similar to other eukaryotic vectors in providing regulatory sequences that control expression of the inserted gene(s).

The majority of vectors are incapable of giving rise to replication-competent viruses since the inserted gene substitutes for a piece of the viral genome that encodes some function essential for propagation of the viral stock. This function can be provided by utilization of a non-defective helper virus that includes the replication and/or structural proteins lacking in the defective virus. Alternatively, a number of packaging cell lines are available that provide the helper functions in trans. Such lines can be used to generate a virus that is fully infectious but cannot replicate and hence cannot be transmitted from cell to cell or cell to host. The type of cell that can be infected is determined by the choice of helper virus or by the nature of the helper functions contained within the packaging cell lines.

Retroviruses can be classified in many ways, but for the purposes of these Guidelines need to be considered according to the cells of the particular species that they can infect. The following definitions are commonly used:

Ecotropic viruses

The ecotropic viruses are those that will grow in cells of the species from which they were isolated and to a limited or undetectable level in cells of other species.

Xenotropic viruses

The xenotropic viruses are endogenous to a species but cannot replicate well in that species, generally because of a receptor block. They tend to have a wide range for replication in cells of heterologous species.

Amphotropic viruses

Amphotropic viruses will grow in the cells of the species from which they were isolated and also in cells from a wide range of other species.

Definitions are taken from:

RNA Tumor Viruses, edited by Robin Weiss, Natalie Teich Harold Varmus, John Coffin, Cold Spring Harbor, 1982.

2.1. In the context of guidelines for handling retroviruses, the major considerations are the nature of the inserted gene and whether the retrovirus has the capacity to infect and propagate itself in human cells. The sequences described below in 2.2 of this Appendix are considered to warrant special attention when incorporated into retroviral vectors that have the capacity to infect human cells.

2.2. The sequences are known oncogenes (of either viral or cellular origin) or any gene encoding molecules that play a role in growth regulation of mammalian cells (for example, growth factors, growth-factor receptors or molecules whose expression is modulated by activation of a growth-factor receptor) and genes encoding toxic or potentially toxic products.

3. Recommendations for Handling Retroviruses

The primary hazard associated with handling retroviruses, particularly xenotropic, amphotropic and human ecotropic retroviruses, is to the manipulator, where the virus might penetrate broken skin or be introduced into tissues by accident (needles or other sharp objects). Potential dangers to other laboratory personnel are much smaller because retroviral particles are extremely labile. Moreover, the risk is further reduced when replication- defective retroviruses are used.

It should be noted that the construction of recombinant retroviruses should be carried out in accordance with Guidelines that govern small-scale laboratory research. Once constructed, the following considerations and practices should be adopted when

handling recombinant retroviruses:

3.1. Rodent and other non-human ecotropic retroviruses are not considered to be associated with any hazard to the manipulator or other laboratory personnel. The practices associated with good tissue-culture technique will be adequate in containing and handling these viruses. While the risks associated with the use of viruses of this group are considered negligible, solutions and contaminated cells should be decontaminated before disposal to prevent accidental infection of other rodent cell lines.

3.2. Procedures for handling defective retroviruses with human cell host range Since the major hazard associated with the use of these viruses is to the operator, the major requirement is for good virological and tissue culture technique on the part of the operator with regard to the following precautions equivalent. Only one individual should use the cabinet at any one time.

3.2.2. Rubber gloves should be worn when manipulating retroviruses containing sequences of the type listed in Section 2 of this Appendix. Also, existing cuts, abrasions and other skin lesions must be protected or covered.

3.2.3. It is recommended that plates of cells containing amphotropic retroviruses be handled in larger plates (or inverted lids) to provide a trap for accidental spills.

3.2.4. All pipettes and glassware should be decontaminated after use by autoclaving or using chlorine disinfectant at the manufacturer's recommended strength. All waste should be autoclaved before disposal.

3.2.5. Mouth pipetting is strictly prohibited.

3.2.6. The use of sharp instruments such as syringes ("sharps") and glass pipettes should be kept to a minimum. Great care should be taken when using "sharps", as skin abrasions represent the most likely point of entry. "Sharps" used when dealing with amphotropic cell lines or viruses should be kept in separate biological-disposal receptacles and sterilized before discarding. These receptacles should not be accessible to other workers.

3.2.7. Tissue cultures infected with ecotropic retroviruses should be kept in separate incubators to those containing cultures infected with amphotropic retroviruses.

3.2.8. Amphotropic virus stocks should be stored in a section of the freezer specifically designated for this purpose and clearly marked to this effect. Similarly, ampoules of frozen amphotropic cell lines should be stored in a separate section of the liquid-nitrogen tank. Laboratory personnel who leave the laboratory should ensure that these materials are either discarded or entrusted to another worker. A central register should be maintained which includes a record of stored cell lines and amphotropic viruses.

This is the responsibility of the IBC.

3.2.9. Great care should be taken to immediately decontaminate spills by washing down contaminated surfaces with dilute bleach (refer to Appendix 9, Clause 8). The correct disinfectant to use in any given situation depends upon the organism being handled, and it is the responsibility of the scientist in charge of the work to select an appropriate disinfectant. In Class II biosafety cabinets, glutaraldehyde disinfectants should be used. After each session, work surfaces should be wiped down in the above manner before ultra-violet sterilization. Where a hood has been used for handling amphotropic retroviruses, subsequent use with non-amphotropic viruses can be undertaken following decontamination with bleach and ultra-violet resterilization.

3.2.10. Only highly trained personnel should be allowed to handle retroviruses capable of infecting human cells. This is the responsibility of the head of the laboratory or chairman of the department in consultation with the IBC.

3.2.11. Under no circumstances should investigators infect cultures of their own cells or of their immediate relatives or of other members of the laboratory.

3.2.12. Only human cells that have been shown to be free of AIDS or other human retroviruses should be used for subsequent infection with recombinant amphotropic retroviruses.

3.2.13. Before beginning work with potentially infectious genetically manipulated retroviruses, investigators are advised to consider lodging a serum sample for subsequent health surveillance.

4. Infection of Animals with Retroviral Vectors

4.1. Non-human ecotropic retroviruses Viruses of this group are not considered hazardous and accordingly good animal-handling practices are appropriate. Infected animals should be kept in separate cages, but can be held in the same room as non-infected animals. All waste generated from animals of this group should be decontaminated before disposal.

4.2. Retroviruses with the capacity to infect human cells Animals infected with retroviruses with a capacity to infect human cells should be kept in a separate cage which is clearly labeled as containing the virus in question. The main risk is to the handler who should take great care to avoid scratches or being bitten. Gloves should be worn to avoid direct contact with tissues and body fluids and benches should be covered with protective paper, which should be replaced regularly. Only highly trained staff should handle these animals under the supervision of the Principal Investigator. Strict precautions should be taken to prevent animals to escape and come into contact with other animals. All waste should be decontaminated before disposal.

APPENDIX 7PROCEDURES FOR WORK WITH HAZARDOUS FRAGMENTS OF DNA

Note for Category 2 in Section 2.2.

When working with isolated DNA molecules, there are some cases where caution is warranted. Some degree of risk may exist and the extent of this is uncertain. Such cases include:

DNA which encodes an active oncogene product, particularly when this is associated with a gene promoter with high activity in human cells. DNA containing more than one active oncogene is associated with increased risk.

DNA encoding growth factors, their receptors or other substances that might directly or indirectly alter the growth patterns of human cells.

DNA or RNA representing complete viral genomes or fragments with the potential to regenerate live virus. Complete genomes for HIV or papilloma viruses, for example, warrant careful handling.

There is some risk that such molecules could enter the cells of the operator, the principal routes of entry being breaks in the skin. It is, therefore, recommended that work of this type be carried out using gloves in order to avoid skin contact. Special care should be taken when using needles or other sharp objects.

Handling retroviruses that include genes encoding oncogenes is dealt with in Appendix 6.

APPENDIX 8

SUPPLEMENTARY INFORMATION REQUIRED FOR WORK WITH WHOLE PLANTS

Classified as Category 2 B (Genetic Manipulation of Whole Plants)

In addition to the proposal forms for small-scale work, supplementary information must be submitted to the IBC in the case of work with whole plants . The following points must be covered:

1. Name of organization. 2. Title of project (as on proposal form for small-scale work). 3. Description of the experimental system to be used (plant species, vector, etc.). 4. Are the experimental plants noxious weeds or closely related to species which

are noxious weeds? If "yes", please elaborate.

5. Are the microorganisms, fungi, etc. involved in this work known to be harmful to man, animals or plants? If "yes", a. give further information about the harmful agent; b. detail the known and likely transmission modes (including carrier insects) for this agent.

6. Are the genetically manipulated plants to be grown? If "yes",

a. what developmental stage will they reach? b. describe the techniques to be employed to contain plant materials (including pollen, seeds, spores, vegetative materials during and at the completion of the experiments; c. what is the proposed method of disposal of plant materials at the conclusion of the experiment?

7. Soil.

a. Is soil or soil substitute to be used (specify)? b. How will it be sterilized?

8. Description of the facility to be used for the cultivation of the plants. Include information such as location, proximity to containment laboratory, etc.

9. Any additional information which may be relevant to the assessment of this work.

10. Signature of the Principal Investigator/Project Supervisor. Date.

The IBC must assess the proposed work on the basis of this supplementary information, and the assessment must be signed by the IBC Chairman.

APPENDIX 9 REQUIREMENTS FOR PHYSICAL CONTAINMENT LEVEL C1

The emphasis in the case of the C1 containment level is essentially on the procedures to be observed by laboratory workers so as to ensure a basic level of laboratory safety. There are also some requirements relating to laboratory design and equipment.

Laboratory Procedures

Many of the laboratory procedures detailed below are those that would be considered essential minimum practices in any microbiological laboratory.

1. Laboratory doors must be closed when work is in progress. 2. Mouth pipetting is prohibited. 3. Eating, drinking, the application of cosmetics and smoking are prohibited. 4. Storage of food or drink in the laboratory or in any storage facility containing

genetically manipulated DNA (e.g., refrigerators) is prohibited. 5. Laboratory coats or gowns must be worn during work and removed before

leaving the laboratory. Suitable coat hooks must be provided within the laboratory and adjacent to the access door.

6. Hands must be washed with liquid soap and warm water when leaving the laboratory and after handling cultures.

7. All microbiological waste must be steam-sterilized before disposal. 8. Equipment used for handling cultures or contaminated material which has not

been steam-sterilized must be disinfected after use. A suitable disinfectant for glassware is a hypochlorite solution containing at least 5,000 parts per million of available chlorine (household bleach diluted 1: 8 is satisfactory and must be made up daily). It must be allowed to act for at least 30 minutes.

9. Work benches and surfaces must be decontaminated with a disinfectant solution after spills and when work is completed.

10. Other disinfectants must be made up and used according to the manufacturer's instructions.

11. Material to be taken from the laboratory to an autoclave, or elsewhere in the building, must be carried in a closed, unbreakable outer container. Provision shall be made to allow for the penetration of steam into the container during autoclaving.

12. All technical procedures must be performed in a way that minimizes the creation of aerosols. In particular, operations such as sonication or vortexing, which may generate aerosols, are to be performed in a biological safety cabinet.

13. All work performed in a C1 laboratory must follow C1 procedures whether or not genetic manipulation work is involved.

14. 14. Essential laboratory equipment includes: o access to a steam-sterilizer in the same building; and o a biological safety cabinet if significant quantities of aerosol are to be

produced.

The biological safety cabinet shall comply with the following Australian Standards:

o A.S. 2251.1 - biological safety cabinets (Class I) for personnel protection; or

o A.S. 2252.2 - laminar-flow biological safety cabinets (Class II) for personnel and product protection.

15. The laboratory must be labeled with adhesive signs (available from the IBC) as follows:

o on the door, with a sign designating the level of containment; o in the laboratory, with a sign listing procedures required for work at this

level of containment.

The signs for laboratories are available from the NBC Secretariat, only after the IBC has certified a laboratory as C1, and only after an NBC inspection: Freezers, refrigerators and other storage units for recombinant or manipulated DNA material must be posted with the universal biohazard symbol, available from distributors of standard laboratory supplies.

APPENDIX 10REQUIREMENTS FOR PHYSICAL CONTAINMENT LEVEL C2

Laboratory Procedures

Work in laboratories at C2 level must conform with the procedures set out for C1 laboratories. In addition, the following requirements apply:

1. All aerosol-producing equipment such as that for sonication and vortexing must be kept and used in the biosafety cabinet.

2. The biosafety cabinet and/or the laboratory must be decontaminated with formaldehyde gas after major spills of contaminated material.

3. No other work is to be done simultaneously with work requiring C2 containment.

4. While work is in progress, a sign on the door must indicate the level of containment required for that work.

5. Protective clothing must not be worn outside the laboratory; it must be transported to the decontamination area in sealed bags or boxes. Boxes must allow penetration of steam during autoclaving.

6. No one may enter the laboratory for cleaning, servicing of equipment, repairs or other activities unless the principal investigator or the biosafety officer has been informed and laboratory surfaces have been disinfected.

Laboratory Planning and Performance Requirements

7. Location.

The Laboratory shall not be located adjacent to, nor open onto corridors used by the general public.

8. Laboratory Planning and Construction.

As detailed below, the laboratory is required to operate at a reduced air pressure. To maintain this pressure, an airlock shall be provided at the entrance.

The airlock shall be fitted with two outward opening doors in series, each fitted with glass viewing panels and automatic door closers. The outer door shall be fitted with a security lock. The structural design of all surfaces of the laboratory, including windows, shall allow for all air pressure loads imposed by the ventilation fans during normal and restricted-inlet operation.

The construction and finish of all of the room surfaces shall be selected to ensure substantially air-tight construction. These surfaces, including those of bench tops and cupboards, shall be smooth, impervious and selected to resist attack by all decontaminating liquids, gases and agents used in the laboratory. Benches, cupboards and engineering services shall be either sealed to the room surfaces or mounted on stand-offs thus permitting wipe-down access for decontamination.

The use of false ceilings and inaccessible spaces should be avoided. Accessible voids such as roof spaces around the laboratory shall be protected against inadvertent access which could cause structural damage or penetration of the barrier.

9. Laboratory Ventilation.

The laboratory shall be maintained at an air pressure of at least 50 pascals below the pressure of adjacent rooms when both doors of the airlock are closed. When either door is open, this pressure should remain at least 25 pascals below that of the adjacent rooms.

The pressure differential shall be achieved by means of an independent room exhaust fan discharging to the outside atmosphere through a filter. A variable speed drive on the exhaust fan is preferred to facilitate manual room pressure control.

Replacement air to the room should be drawn through a filtered aperture which is adjustable to assist in setting up the reduced room pressure. The replacement air filter shall be a Type 1 Class A or Class B complying with A.S. 1324 and having a minimum arrestance efficiency of 90 percent when tested in accordance with A.S. 1132.5 with Test Dust No. 2.

The exhaust filter shall be a HEPA type as specified in A.S. 1324 clause 4.3.1 (b). This filter shall be fitted with a prefilter having the same specifications as the replacement-air filter. The HEPA filter shall have metal separators. Fluid or grease seals shall not be used. Access shall be provided to facilitate determination of the integrity of the HEPA filter installation in accordance with A.S. 1807.6. Means shall be provided to monitor pressure drop across the prefilter.

A magnetic type differential-pressure gauge shall be provided within the laboratory to indicate negative room pressure. Other air conditioning control switches and the exhaust fan speed-setpoint control should be located adjacent to the gauge. An audible alarm to indicate loss of room pressure should be provided.

10. Supplementary Cooling.

Where the exhaust ventilation rate is inadequate to offset room heat loads, supplementary cooling in the form of a fan coil unit may be used, utilizing chilled water or refrigerant as the cooling medium. Particular attention should be paid to the positioning of this unit in the room in order to avoid airflows likely to disturb the operation of the biosafety cabinet.

11. Biosafety Cabinet.

A biosafety cabinet of the type specified for C 1 containment shall be provided. Installation of the cabinet shall comply with the requirements and recommendations of A.S. 2647 (Biological Safety Cabinets: Installation and Use). This standard also makes reference to airflow disturbances in front of biological safety cabinets.

12. Decontamination.

Provision shall be made to decontaminate the biological safety cabinet (s) and the room independently with formaldehyde gas, and for the gas to be purged safely to atmosphere upon completion of the procedure.

Decontamination of the safety cabinet (s) should be performed in accordance with the requirements of A.S. 2547 Appendix C and will require the provision of a front cover plate and exhaust duct adapter to fit the particular cabinet.

Decontamination of the room will require a close-off damper in the discharge from the exhaust filter and means of closing the room replacement- air aperture. Remotely switched power points facilitate the safe generation of the formaldehyde gas.

13. Steam Sterilizer.

The laboratory shall have close access to a steam sterilizer.

14. Pest Control Program.

A pest control program against insects, rodents, birds, possums, etc. shall be instituted.

15. Signs.

The laboratory must be labelled on the door and wall with C2 signs. These signs are available from the NBC Secretariat only after NBC has inspected the laboratory and certified it for C2 containment.

APPENDIX 11 REQUIREMENTS FOR PHYSICAL CONTAINMENT LEVEL C3

The requirements for a C3 laboratory are much more stringent than for a C2 laboratory. Air supply and drainage facilities must be separate from those of the rest of the building, and various items of equipment must be installed inside the laboratory.

Institutions planning C3 facilities are asked to liaise with NBC.

APPENDIX 12REQUIREMENTS FOR PLANT-HOUSE LEVEL PH1

The following standard is regarded as a suitable minimum for genetic manipulation work with whole plants which falls under Category 2 in Section B. Most work with plants will be adequately contained in such plant houses.

Other plant-house work may require at least the PH1 level of containment, and additional operating procedures and/or a higher standard of construction may be recommended.

Plant-House Level PH1

Construction

1. The plant house must have a concrete floor, any openings in the walls or roof (e.g., windows, vents) must be screened with fine screens (thirty-gauge 30/32 mesh wire gauze). The drainage exits must be designed to avoid entry of rodents and insects.

2. Entrances to the plant house must be posted with a sign identifying the type of plant house and listing the procedures applicable, including emergency and maintenance procedures.

3. If the plant house is free-standing, it must have an anteroom for entry and exit. The anteroom must be fitted with a sticky pest strip or automatic insecticide aerosol device designed to kill arthropods which gain entry. An anteroom is not necessary if the plant house connects directly with a certified containment facility for small-scale or large-scale work.

Operating Procedures

4. The plant house must be inspected regularly to ensure that its containment features are intact. Screens, filters and the like must be cleaned regularly (in accordance with manufacturer's specifications when provided).

5. All doors to the plant house must be locked for the duration of the work except for those periods when personnel are actually working inside the plant house.

6. Hands must be washed with soap and water before leaving the plant house. 7. Only persons authorized by the IBC are to enter the plant house. All such

persons must be trained to follow normal plant-house routines as well as these operating procedures.

8. All plants in the plant house must be treated as plants containing genetically manipulated DNA. Work in the plant house other than that involving genetic manipulation should be discouraged.

9. Operations which may generate aerosols are to be performed in a biological safety cabinet as specified for C1 containment.

10. Plants and tissues taken into or out of the plant house must be carried in closed containers. Waste plants, tissues, soil, soil substitutes and the containers must be

sterilized. 11. Living plants or tissues must not be taken from the plant house except to a

containment laboratory or, with the approval of the IBC, when they are being transferred to another organization.

12. If the work permits, plants should be sprayed regularly with a systemic insecticide. The plant house must be sprayed or fumigated to kill other arthropods (especially mites) at regular intervals and at the end of each series of experiments. The organization must have an effective insect and rodent control program.

13. The experimental materials must be inspected regularly for signs of arthropod infestation. The inspection regimen must pay particular attention to mites as they would not normally be excluded by the window and vent screens.

PH1 signs are available from the NBC Secretariat after IBC certification.

APPENDIX 13NOTE ON PLANT HOUSES WITH HIGHER CONTAINMENT THAN PH1

Certain experiments may warrant the use of plant houses with additional containment features and operating procedures resulting in a containment level higher than PH1 (Appendix 12). As the necessary measures are likely to vary depending upon the nature of the hazard and the medium by which it is normally dispersed, NBC has decided that it is not practical to set discrete specifications for higher-containment plant houses. Each project will be considered on an individual basis, and in many cases NBC may recommend additional operating procedures to be used with a PH1 facility, rather than recommend PH2. The following requirements are recommended for institutions which may be planning to build plant houses providing containment greater than PH1.

The notes in this Appendix and in Appendix 14 are provided so that IBCs can identify some of the requirements which need to be incorporated into the design of PH2 and PH3 containment facilities.

PH2 door signs will be provided by NBC after NBC inspection and certification.

REQUIREMENTS FOR PLANT-HOUSE LEVEL PH2

The PH2 standard must meet all the requirements of the PH1 standard as well as those listed below. Note that references to plants or plant tissues in PH1 should be regarded as referring to all organisms when the PH2 standard is being considered.

Construction

1. The joins between any structural components must be sealed. Transparent sections should preferably be made of impact-resistant material such as methyl- methacrylate ("Perspex") or reinforced glass. If ordinary glass is used, a hailstone screen must be fitted.

2. Air supply and exhaust ducts must be fitted with fine screens (thirty-gauge

30/32 mesh wire gauze). 3. If the plant house is free-standing, it must have an anteroom for entry and exit.

The innermost door must have a door-closing device. The anteroom must be fitted with a sticky pest strip or automatic insecticide aerosol device designed to kill arthropods which gain entry. An anteroom is not necessary if the plant house connects directly with a certified containment facility for small- scale or large-scale work.

4. A wash basin must be located either within the anteroom or in the plant house close to the entry. Where a laboratory is directly connected to the plant house, the basin may be in the laboratory.

Operating Procedures

5. When entering and leaving the plant house, personnel must decontaminate their hands by washing with soap and warm water in the wash basin provided. When entering, personnel must put on overshoes, covering clothes (e.g., gown/boiler suit) and a hat in the anteroom. These overshoes and clothes must be removed when leaving the plant house and kept in the anteroom (or laboratory) between uses. They must be laundered regularly.

6. Materials and equipment taken into or out of the plant house must be treated by a technique demonstrated to be effective in destroying or removing all stages of the life cycle of arthropods. This requirement applies to soil substitutes and, where feasible, to soil. Soil substitutes which can be readily decontaminated should be used whenever possible. Use of soil is discouraged.

APPENDIX 14REQUIREMENTS FOR PLANT-HOUSE LEVEL PH3

See note on plant houses with higher containment than PH1 in Appendix 13.

The PH3 standard must meet all the requirements of the PH1 and PH2 standards as well as the following:

Construction

1. The transparent sections must be constructed of impact-resistant materials. Ordinary window glass is not sufficient irrespective of whether it is proposed to install hail screens.

2. All drains in the plant house and anteroom must empty into collecting tanks. The floor of the plant house, the lower parts of the walls and the sills under doors must be constructed and sealed to ensure that water spillages drain only into the collecting tanks.

3. Sealing of the plant house and anteroom must be sufficient to enable decontamination by gas.

4. The anteroom must contain an autoclave, and the wash basin must be fitted with foot- or elbow-operated taps.

5. The air pressure in the plant house must be maintained at a level 50 Pa below the external air pressure. There must be a meter showing the pressure differential and an alarm which operates when this differential is not maintained. Air-supply and air-exhaust ducts must be fitted with HEPA filters.

6. Mechanical equipment (e.g., air-supply and air-exhaust units, water pumps) must be located outside the facility to allow for repair and maintenance without entry into the facility. If this equipment may contain or process materials from inside the plant house, it must be designed to prevent the escape of viable organisms or be located in a sealed housing which can contain any leaks and be decontaminated if required. (In the event of a power failure, no entry into the plant house is permitted until services have been restored.)

Operating Procedures

7. Except where the experimental organism(s) is a microorganism, all materials and equipment brought into the plant house must be treated to kill microorganisms.

8. Footbaths (metal trays containing cloth pads soaked in a disinfectant) must be located in the anteroom adjacent to the internal plant-house door.

9. Plant containers must be located in or over water-tight trays which drain directly into the drainage system.

10. Before waste water is discharged from the collecting tanks, it must be decontaminated in a way which will destroy microorganisms as well as plant and arthropod material.

11. Work surfaces and the floors must be decontaminated regularly and immediately after spillages. The plant house and anteroom must be fumigated against microorganisms at the end of each series of experiments.

APPENDIX 15REQUIREMENTS FOR EXPERIMENTS INVOLVING TRANSGENIC ANIMALS

Introduction

Genetic manipulation work which involves the introduction of DNA into the fertilized oocyte or early embryo, or which may be carried out in or involve whole animals, falls under Category 2.

However, proposals which fall under Category 1 (i.e., the introduction of a fragment of the whole genome or a virus into the fertilized oocyte or early embryo), require a recommendation from the NBC.

Essential References

All investigators and IBCs responsible for genetic manipulation work which involves

transgenic animals should be familiar with the requirements of:

Code of Practice for the Care and Use of Animals for Experimental Purposes (NH&MRC/CSIRO/Australian Agricultural Council 1989).

Australian Standard 2243.3/1985: Safety in Laboratories, Part 3: Microbiology (basic standards for animal houses).

Procedures for Assessment of Planned Releases of Recombinant DNA Organisms, May 1987, available from the GMAC Secretariat, PO Box 2183, Canberra 2601.

Other useful references are given in Appendix 1 of these Guidelines.

Procedures

Principal Investigators.

Investigators planning work which will fall under Category 2 (A) (i.e., production of transgenic animals) and which will not fall under Category 1 nor involve planned releases of genetically manipulated organisms should:

1. Complete a proposal form for small-scale work.. 2. Attach a description addressing each of the points listed in Appendix 17 (1- 8). 3. Submit the proposal to their IBC. 4. Not start work on those aspects which involve animals until IBC approval has

been received.

Institutional Biosafety Committees.

Upon receipt of a proposal for work with transgenic animals, the IBC should:

1. Assess the information provided in the proposal form and the description of the animal facilities.

2. Inspect the proposed facilities. 3. Either (i) approve the proposed protocol and facilities, or (ii) recommend

additional precautions and give approval when these have been implemented. 4. Attach a copy of the terms of the approval (which may be included in Section 8

of the IBC assessment form) if IBC approval is qualified in any way, or if additional conditions are required, and provide this advice to the investigator.

5. Ensure that the experimental protocol has been approved by an institutional ethics committee.

6. Notify NBC of the IBC certification of the transgenic-animal holding facilities, and send a copy of the assessment and proposal forms to NBC for notification, including IBC advice.

APPENDIX 16REQUIREMENTS FOR HOUSING OF INFECTIOUS TRANSGENIC ANIMALS

Points to consider for the housing of animals in which infectious agents have been used, including transgenic animals produced by infectious agents.

1. The animal housing must comply with the &Code of Practice for the Care and Use of Animals for Experimental Purposes" (NH and MRC/CSIRO/AAC, Australian Government Printing Services, 1989).

2. Laboratory animal facilities are an extension of the laboratory and may be integral to, and inseparable from, the laboratory.

3. As a general principle, the biological and physical containment recommended for working with infectious agents in vivo and in vitro are comparable.

4. The physical containment levels for work with infectious genetically manipulated animals follow the animal containment levels C1, C2 or C3 as appropriate for the pathogen. Requirements for C1 and C2 animal containment facilities are set out in Appendices 18 and 19. Animal facilities associated with C3 containment require special additional considerations, and NBC advice should be sought on these.

APPENDIX 17DESIGN OF TRANSGENIC-ANIMAL FACILITIES

Points for IBCs to consider in the design of facilities for genetic manipulation work involving the production of transgenic animals.

Note on Animal Housing

Facilities for laboratory animals used for studies for genetic manipulation work of either an infectious or non-infectious nature should be physically separated from other activities such as animal production and animal quarantine.

Animals under experiment may be either small laboratory animals (e.g., mice, rabbits) or large domestic animals (e.g., pigs, sheep, cattle). The requirements for the housing and maintenance of the animals may differ in scale as a result, but the microbiological safety principles are similar.

The particular point of concern with transgenic animals is that the result of any insertion of novel genetic material into the genome of an animal is unpredictable, both in terms of the expression of the new genetic material and regarding the effect of this material on neighbouring genes. Furthermore, any escape of the novel genetic material through either the escape of transgenic animals or through their interbreeding with wild or uncontained animals is potentially irreversible.

The containment required for transgenic animals varies among different experimental

systems. The following points govern the nature of containment involved:

Details of Animal Involved.

1. Class, genus and species of animal (if transgenic invertebrates or aquatic vertebrates are involved) (Appendix 15).

2. Stages in life cycle that will be involved (embryo only to full maturity and reproduction).

3. Approximate number of animals involved at any one time. (If large numbers of transgenic animals are involved, NBC advice may be required.)

Type of Work.

4. Whether the work will involve:

(a) modification of germline cells; (b) modification of somatic cells; (c) intention to breed; (d) infectious agents used to produce transgenic animals (in which case the proposal requires NBC review).

Details of the Genetic Manipulation.

5. Depending on the type of work, the following points should be considered before deciding upon the level of animal containment (C1, C2 & C3):

(i) nature of DNA to be inserted (origin and characterization of DNA, references to published work, reference numbers of small-scale proposals covering preparation of the DNA); (ii) method to be used for introducing the DNA (including details of the construction of any vector to be used, references to published work or reference numbers of small-scale proposals covering preparation of the vector, and containment level recommended for work with the vector); (iii) likelihood of the technique to be used for introducing the DNA resulting in the animal bearing an agent potentially transmissible to animals in the same facility or to humans, and the possible routes of transmission; (iv) reproductive capability of the genetically manipulated animal - is the inserted DNA likely to be heritable?

Other Work Being Performed in the Same Holding Facility.

6. Are there any other animals being housed in the same facility? If so,

(i) how many animals are being housed? (ii) are they all the same species and strain? (what other species are involved?)

(iii) what is the nature of any experimental work being carried out on the other animals (for example, infectious-disease work, or genetic manipulation work)? Is it likely that these experiments will interfere with the proposed work? (iv) what arrangements are there to ensure that the other animals are kept separate from the transgenic animals? (v) what procedures are in place for the identification of, and accounting for, individual animals?

Disposal of Animals.

7. Bearing in mind the points to consider and the information provided above, consider what special precautions are needed, in addition to standard animal- house practices, regarding the disposal of animals.

Transport of Animals.

8. If it is necessary to transport the animals alive from the holding facility to the laboratory or another facility, consider what precautions are needed to be taken in addition to the provisions set out in Chapter 4 (Section 3) of the Guidelines.

APPENDIX 18 REQUIREMENTS FOR ANIMAL-CONTAINMENT LEVEL C1A

Construction 1. Entrance to the animal house must be posted with a sign identifying the type of

animals house and listing applicable procedures, including emergency and maintenance procedures.

2. The animal room must be constructed with impermeable and easily cleanable surfaces. Any openings in the walls or roof (e.g., windows or vents) must be screened with fine screens (60 x 40 swg mesh - 51 % free area). The doorway and room structure should be rodent-proof. Drainage exits in the floor should always contain water or disinfectant in the trap.

3. Doors to animal rooms should open inwards and be self-closing and should be kept closed when experimental animals are present and for those periods when work is being carried out within the room. If the animal house connects directly with a large-scale containment facility, there may be a requirement for an anteroom. If the animal house is separated from the containment laboratories, an anteroom in which protective clothing and footwear can be stored is required.

Operating Procedures

4. The animal facility must be inspected regularly by the IBC to ensure that its containment features are intact. Only people authorized by the IBC can enter the animal house. All such people should be trained in normal animal-house procedures as well as these operating procedures. A record book should be maintained to provide an up-to-date inventory of the procedures performed.

5. Standard procedures for containment at C1 level must be followed. Work

surfaces are to be decontaminated after use and after any spill of viable material. Eating, drinking, smoking and the storage of food for human use is not permitted in animal rooms. Personnel should wash their hands after handling cultures and animals and before leaving the animal room.

6. Operations which may generate aerosols are to be carried out in a biological safety cabinet as specified for C 1 containment.

7. Protective clothing, gloves and footwear should be worn. It is further recommended that such footwear and clothing not be worn in other areas. Protection against inhalation of aerosols, scratches or bites should be considered.

8. Bedding material and waste from animal cages or pens should be removed in such a manner as to minimize the creation of aerosols. This material should be rendered safe by regular sterilization.

9. Special attention should be paid to constraining animals during experiments. The prevention of their escape needs to be ensured.

10. Animals or animal tissue transported in or out of the animal house must be carried in closed containers. Animals involved in genetic manipulation experiments are not to be used for other purposes or to provide tissue for other purposes. Live animals or animal tissue taken from the animal house must only go to another containment facility or be transferred to another organization which has suitable animal containment facilities. (See Chapter 4, Section 3, or requirements regarding the transfer of infectious materials.)

11. Animal carcasses must be rendered safe by sterilization before disposal. 12. If genetic manipulation experiments are conducted on invertebrates or aquatic

vertebrates, the following additional conditions apply:

(i) a mechanism should be provided to ensure that neither the organisms nor their gametes can escape into the supply or discharge system of the rearing container (e.g., tank or aquarium); the top of the rearing container should be covered to avoid escape of organisms and their gametes; (ii) in the case of invertebrates that crawl, jump or fly, manipulated and non-manipulated animals should be kept apart; (iii) measures should be taken to enable escaped invertebrates to be detected and recaptured or destroyed; for ticks and mites, containers should be kept over trays filled with oil; (iv) all experimental cages/pens must be numbered and documented; (v) used culture vessels must be decontaminated before disposal or thoroughly cleaned before reuse; (vi) flying or crawling arthropods should be handled on white trays to facilitate the detection of escape; (vii) the use of an electric insect-control unit should be considered; (viii) the activity of arthropods and the risk of accidental escape can be reduced by chilling.

It should be borne in mind that invertebrates can harbour infectious organisms, for example, viruses in mosquitoes, midges and biting flies in soft ticks, trypanosomes in triatomid bugs, organisms from sewage in mollusca, crustacea or echinodermata. Such

invertebrates should be kept at a containment level appropriate to the risk from the human pathogens carried by these invertebrates.

APPENDIX 19 REQUIREMENTS FOR ANIMAL-CONTAINMENT LEVEL C2A

Similar constraints to those applying to C2 laboratories also apply to animal facilities at C2A level.

Construction

1. Entrance into C2A animal facilities is limited to people aware of the potential hazards. The entrance door should be fitted with a "fire-escape lock" (i.e., it should be accessible from outside using a special key but freely accessible from inside). The door must be posted with signs identifying the facility and listing the applicable procedures. The name and telephone number of the animal-facility supervisor or another responsible person should be attached.

2. The C2A facility must not be accessible to the general public or open onto a public thoroughfare.

3. The C2A facility must be isolated from other animal-facility areas by an airlock having two doors in series, each fitted with automatic closers. The C2A area must be maintained at a minimum negative pressure of 50 Pa when both doors are closed and 25 Pa when one door is open. The negative pressure must be achieved by means of an independent room exhaust discharging to open air through a filter. The replacement air must be drawn into the room through a filtered aperture the size of which can be adjusted and set to achieve the required negative pressure.

All filters must be of medium grade having a minimum efficiency to arrest 95 % of all particles above 5 micrometers. To prolong the life of the exhaust filters by protecting them against clogging by animal hair and feed dust or, in the case of poultry, down and skin dander, a roughing filter should be used preceding the main filter. This will also enable filter changing procedures in which one filter is always interposed between the room and the outside atmosphere.

Ventilation rates should ensure an acceptable atmosphere quality for animal welfare. If air cooling is required, this should be achieved by individual "spit" type air conditioners which do not require direct exchanges of air.

4. The facility must be constructed so that the finishes on walls, floors, ceilings and benches are impervious and easy to clean.

5. All waste from the animal facility must be decontaminated by autoclaving prior to disposal. If floor drains are present, the drain traps should always be filled with water or a suitable disinfectant. When animals are in the room, floor drains should be sealed with an airtight plug.

6. Pest-control systems for insects, rodents, etc. must be instituted. 7. It should be possible to decontaminate the C2A area independently with

formaldehyde gas and for the gas to be discharged safely to the atmosphere upon completion.

8. The facility must have access to an autoclave either within the C2A area or in the animal building.

9. The facility must have a hand basin and foot and elbow operated taps located near the exit.

10. Biological safety cabinets are to be used within the C2A facility for containment of aerosols and control of infectious material.

Operating Procedures

11. Standard procedures for C2 containment level must be followed. No other work is to be performed simultaneously with work requiring C2A containment. The C2A facility must be inspected at least annually by the IBC to ensure that its containment requirements are intact. Only people authorized by the IBC are to enter the animal facility and they can do so only after they have been advised of the potential hazards and meet any specific requirements (e.g., immunization). A record should be maintained to provide an up-to-date inventory of the animals present and a chronological record of procedures performed.

12. Protective clothing, gloves and footwear should be worn. Protection against aerosols, scratches and bites should be considered. The protective clothing and footwear must be removed before leaving the facility. Dirty clothing must be decontaminated, preferably autoclaved, before being laundered.

13. Eating, drinking, smoking and the storing of food for human use are not permitted in animal rooms.

14. Personnel should wash their hands after handling cultures and animals and before leaving the animal room.

15. Work surfaces should be decontaminate after use or spills of viable materials. 16. Bedding material and wastes from animal cages/pens should be removed in such

a manner as to minimize the creation of aerosols. This material must be autoclaved before removal from the facility.

17. Cages should be decontaminated by autoclaving before cleaning and washing. 18. Special attention must be paid to containing animals during experiments and to

the prevention of their escape. 19. Live animals must not leave the facility. Animal carcasses must be rendered safe

by sterilization before disposal. Animals used in genetic manipulation experiments are not to be used for other purposes or to provide tissue for other purposes.

Note: NBC inspection and certification is required for animal-containment level C2A.

APPENDIX 20 NOTE ON TRANSPORT

The transport of genetically manipulated organisms, DNA preparations, wastes or by-products (whether containing viable organisms or not) which may be infectious or potentially hazardous must be performed with extreme care.

Regulations: Thailand: Full Text