-
for Contained Use of Genetically Modified Microorganisms
at Pilot and Industrial Scales
TECHNICAL BIOSAFETY COMMITTEE (TBC)NATIONAL CENTER FOR GENETIC
ENGINEERING AND BIOTECHNOLOGY (BIOTEC)
NATIONAL SCIENCE AND TECHNOLOGY DEVELOPMENT AGENCY (NSTDA)
MINISTRY OF SCIENCE AND TECHNOLOGY (MOST)
Biosafety Guidelines
2015
-
Biosafety Guidelines
for Contained Use of Genetically Modified Microorganisms
at Pilot and Industrial Scales
TECHNICAL BIOSAFETY COMMITTEE (TBC)
NATIONAL CENTER FOR GENETIC ENGINEERING AND BIOTECHNOLOGY
(BIOTEC)
NATIONAL SCIENCE AND TECHNOLOGY DEVELOPMENT AGENCY (NSTDA)
MINISTRY OF SCIENCE AND TECHNOLOGY (MOST)
2015
-
Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
Technical Biosafety Committee
National Center for Genetic Engineering and Biotechnology
National Science and Technology Development Agency (NSTDA)
© National Center for Genetic Engineering and Biotechnology
2015
ISBN : 978-616-12-0386-3
Tel : +66(0)2-564-6700
Fax : +66(0)2-564-6703
E-mail : [email protected]
URL : http://www.biotec.or.th
Printing House : P.A. Living Printing Co.,Ltd
4 Soi Sirintron 7 Road Sirintron
District Bangplad Province Bangkok 10700
Tel : +66(0)2-881 9890
Fax : +66(0)2-881 9894
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Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales i
Preface
Genetically Modified Microorganisms (GMMs) were first used in
B.E. 2525 to
produce insulin in industrial medicine. Currently, GMMs are used
in various industries,
such as the food, pharmaceutical and bioplastic industries, to
manufacture a number
of important consumer products. To ensure operator and
environmental safety,
the Technical Biosafety Committee (TBC) of the National Center
for Genetic Engineering
and Biotechnology (BIOTEC), the National Science and Technology
Development Agency
(NSTDA), has prepared guidelines for GMM work, publishing
“Biosafety Guidelines for
Contained Use of Genetically Modified Microorganisms at Pilot
and Industrial Scales”
in B.E. 2547. The guidelines have been updated every two years
to take into account
the latest information and technology. In B.E. 2558, GMM waste
management
guidelines were added to facilitate operator work, the list of
microorganisms/agents
was updated to conform to lists of national and international
organizations, and
an English version was prepared for foreign
organizations/institutions involved in
GMM work at pilot and industrial scales in Thailand.
The principle and scope of these guidelines cover the use of
GMMs in
containment at pilot and industrial scales according to GMM
classification, together
with suggested containment levels, GMM waste management,
transport, possession,
emergency plans and the responsibilities of personnel associated
with GMM work.
The committee acknowledges the Biosafety Sub-Committee on
Microorganisms
and the Organizing Committee on Biosafety Guidelines for
Contained Use of Genetically
Modified Microorganism (English version) for their cooperation
and revision of these
guidelines, and Ajinomoto Co., Ltd. for supporting the
preliminary translation.
Finally, the committee hopes that these guidelines will be
helpful in promoting
safe GMM work at pilot and industrial scales. Suggestions and
comments on the
guidelines are most welcome.
(Dr. Kanyawim Kirtikara)
Executive Director
National Center for Genetic Engineering and Biotechnology
Chair
Technical Biosafety Committee
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ii Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
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Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales iii
Contents
Topic Page
Preface
.............................................................................................................
i
Definitions
........................................................................................................
vii
Abbreviations
...................................................................................................
xi
Chapter 1 Introduction
...................................................................................
1
Chapter 2 Scope and Principles
......................................................................
3
Chapter 3 Classification of GMM Work at Pilot and Industrial
Scales ........... 5
Chapter 4 GMM Containment Levels for Pilot and Industrial Uses
............... 9
Chapter 5 Approval Process for Projects with Contained Use of
GMMs at Pilot
and Industrial Scales
......................................................................
15
Chapter 6 Risk Assessment for Contained Use of GMMs at Pilot and
Industrial
Scales
.............................................................................................
17
Chapter 7 Safety Management System for Contained Use of GMMs at
Pilot
and Industrial Scales
......................................................................
19
Chapter 8 Waste Management of GMMs
...................................................... 23
Chapter 9 Emergency Plan and Inactivation of Spilled GMMs in
Contained
Use at Pilot and Industrial Scales
.................................................. 29
Chapter 10 Possession, Transport, Import and Export of GMMs
.................... 31
Appendices
......................................................................................................
37
Credits
.............................................................................................................
161
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iv Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
List of Tables
Table Page
Table 3.1 Summary of GMM work at pilot and industrial scales
............... 8
Table 8.1 Examples of biological indicators for verification of
heat and
chemical inactivation
.................................................................
24
Table 8.2 Waste management requirements
.............................................. 27
Table A1.1 Examples of microorganisms/agents capable of natural
DNA transfer
within the same sublist
...............................................................
38
Table A2.1 Suggested criteria for GILSP
....................................................... 42
Table A7.1 Levels of safety control and safety protective
measures for using
GMMs at pilot and industrial scales
............................................ 133
Table A10.1 Minimum recommended values for inactivating
microorganisms/
agents and waste decontamination cycles
.................................. 147
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Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales v
List of Figures
Figure Page
Figure 5.1 The approval process flowchart for projects with
contained use of
GMMs at pilot and industrial scales
............................................ 16
Figure 10.1 Example of the triple packaging system for the
packaging and
labeling of Category A infectious substances
............................... 33
Figure 10.2 Example of the triple packaging system for the
packaging and
labeling of Category B infectious substances
............................... 34
Figure 10.3 Example of the triple packaging system for GMMs
(GILSP/class 1) 35
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vi Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
List of Appendices
Appendix Page
Appendix 1 Non-Genetically Modified Microorganisms
.............................. 37
Appendix 2 Elaboration of criteria for GILSP (Good Industrial
Large Scale
Practice) GMMs
.........................................................................
39
Appendix 3 List of safe host systems
............................................................ 43
Appendix 4 Classification of human etiologic agents on the basis
of hazard 57
Appendix 5 Examples of human toxins
......................................................... 129
Appendix 6 Basic working procedures for contained use of GMMs at
pilot
and industrial scales for health and environmental safety
........ 131
Appendix 7 Containment for work using GMMs at pilot and
industrial scales
(Large-scale Containment Level, LS)
......................................... 133
Appendix 8 Application form for contained use of GMMs at pilot
and
industrial scales
........................................................................
137
Appendix 9 Criteria for risk assessment of contained use of GMMs
at pilot
and industrial scale (for class 2 GMMs or higher)
.................... 143
Appendix 10 Autoclave parameters for waste treatment
............................... 147
Appendix 11 Sample incident report form
...................................................... 149
Appendix 12 List of related laws, regulations and ministry
notifications ........ 153
Appendix 13 Examples of infectious substances classified as
Category A ...... 155
Appendix 14 References
.................................................................................
157
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Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales vii
Definitions
Bacteriophage: An obligate intracellular virus that multiplies
inside bacteria.
Biosafety level: The level of biosafety of work using GMMs by
implementation
of a containment level. In some countries, biosafety level is
equivalent to ‘containment
level’.
Closed system: A system which separates GMMs from the
environment during
the culturing process, such as a bioreactor or biological safety
cabinet (tissue culture
hood). It also includes production processes utilizing equipment
connected in a closed
system, such as inoculation of GMMs into a bioreactor, and
downstream processes
for product purification, as well as systems where equipment are
not connected but
are set up within a safety enclosure. A closed system used for
GMM activities at pilot
and industrial scales should be routinely checked.
Contained use: The use of GMMs in a restricted area, isolated
from the
outside environment through the provision of tools and
equipment, working space
and working protocols for the purpose of research or industrial
production.
Containment and containment level: Control of GMMs to a
restricted area,
isolated from the outside environment through the provision of
tools and equipment,
working space and working protocols to facilitate research or
industrial production.
There are 4 containment levels which have been classified
according to degree of
risk in terms of human pathogenicity and potential hazard to the
environment.
Controlled area: An area for conducting GMM work such as
inoculation and
propagation of GMMs in a bioreactor, sampling or transport of
GMMs, and
downstream processes such as the purification of GMM
products.
Donor organism: A living organism that is the origin of the DNA
or gene
inserted into a host cell for a desired phenotype.
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viii Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
Genetic modification technique:
1. The use of recombinant DNA technology to ligate DNA fragments
or
heterologous genes of interest with vectors followed by
transformation into
host cells by methods such as electroporation to enable such
host cells to exhibit
desired phenotypes. Plasmids and viruses are examples of the
vectors used.
2. Introduction of DNA fragments or genes of interest into host
cells via
micro-injection, macro-injection or micro-encapsulation.
3. Cell or protoplast fusion and hybridization techniques
between different
cell types with different genetic materials, which produce
heterologous genes in
microorganisms/agents in a manner which cannot occur in
nature.
Genetically Modified Microorganism (GMM): A microorganism/agent
whose
genes or genetic material have been modified from its original
counterpart in
a manner that cannot occur in nature through genetic
modification techniques for
expression of desired phenotypes such as enzyme production. They
include progeny
of such microorganisms, which have inherited the modified
genetic material.
Genetically Modified Organism (GMO): An organism whose genetic
material
has been altered using modern biotechnology.
Good Industrial Large Scale Practice (GILSP): Application of
good
microbiological practice in the use of harmless
microorganisms/agents in industry.
Such microorganisms/agents include non-pathogenic
microorganisms/agents and
GMMs that have a long history of safe use in industry or limited
survival in the natural
environment. Viruses, phages or plasmids that may cause disease
are not used.
HEPA filter: A high efficiency particulate air filter which can
prevent the
passage of small particles under 0.3 micrometers (µm) in size at
99.97% efficiency.
Microorganisms cannot pass through this type of filter.
Host or recipient cells: A cell that has incorporated modified
DNA fragments
or genes for expression of desired phenotypes.
Inserted DNA: Heterologous DNA or gene that is introduced into a
host cell
by a vector or other genetic modification techniques to create
desired phenotypes.
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Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales ix
Institutional Biosafety Committee (IBC): A committee
commissioned by
an institution or organization to provide advice and monitor
work or projects related
to modern biotechnology or genetic engineering according to
biosafety guidelines.
LD50: The amount of a chemical or biochemical substance that
causes death
among 50% of test animals.
Microbial inactivation: The inactivation of GMMs from materials,
equipment,
tools, bioreactors and surfaces which may be contaminated with
GMMs by using an
appropriate procedure, such as heating or chemical treatment, in
a manner that is
not harmful to humans or the environment.
Microorganism: A small living cell or particle that is able to
reproduce and
transfer genetic material. It includes bacteria, yeasts, molds,
viruses, viroids, cultivated
plant cells and cultivated animal cells.
Operator: A person involved in GMM work within an
organization/institution.
Organization and institution: An organization where GMMs are
used for
commercial purposes at pilot and industrial scales, such as
state enterprises,
independent research institutes, factories and private
companies.
Owner or authorized representative: A person who is the head or
designated
representative of an organization and institute.
Primary containment equipment: Equipment that is designed to
provide
containment or eliminate exposure to biohazardous materials,
such as a biosafety
cabinet or an isolater.
Recombinant DNA molecule:
1. Molecules constructed outside living cells by joining natural
or synthetic
DNA fragments to DNA molecules that can replicate in a living
cell, or
2. Molecules that result from the replication of those described
above.
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x Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales
Risk assessment: An analytical process used for assessing risks
posed to
the environment or human health by GMM-related activities. Risks
include direct
and indirect risks, and those with immediate, delayed or
downstream effects.
Technical Biosafety Committee (TBC): A committee whose main
responsibilities
are :
1. To provide technical consultation to any work or project
related to modern
biotechnology or genetic engineering according to biosafety
guidelines;
2. To identify risk categories for activities that are not
clearly classified;
3. To coordinate with agencies responsible for monitoring GMOs;
and
4. To enhance the efficiency of IBCs at the national level.
The use of Genetically Modified Microorganisms (GMMs) in pilot
plants
and the industry: Includes the production of GMMs at a
substantial scale (more than
10 liters) to produce biological substances in contained
conditions with no intention
to release GMMs into the environment.
Vector: DNA capable of self-replication in a living organism,
used for
introducing DNA or genes of interest into a host cell by
ligation to such DNA. Examples
include plasmids and viruses.
Viroid: An infectious agent affecting living cells, smaller than
a virus and
consisting only of nucleic acid without a protein coat.
Virus: A very small agent that cannot reproduce by itself but
must replicate
inside a living cell. One of its prominent characteristics is
that it consists of either
DNA or RNA but not both. Most antibacterials and antifungals
have no effect on
viruses even when used at concentrations that normally inhibit
the growth of bacteria
or fungi.
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Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales xi
Abbreviations
BIOTEC National Center for Genetic Engineering and
Biotechnology
FDA Food and Drug Administration
GILSP Good Industrial Large Scale Practice
GMM Genetically Modified Microorganism
GMO Genetically Modified Organism
IBC Institutional Biosafety Committee
MOPH Ministry of Public Health
NIH National Institute of Health of Thailand
OECD The Organisation for Economic Co-operation and
Development
ONEP Office of Natural Resources and Environmental Policy and
Planning
TBC Technical Biosafety Committee
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xii Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
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Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 1
Chapter 1Introduction
Modernbiotechnologyhasmadegreatstrides,particularly in
thefieldof
recombinantDNA technologywheregeneticmodification
techniquesorgenetic
engineeringareemployedtomodifyorintroduceDNAfragmentsorgenesthatcarry
desiredcharacteristicstolivingorganismssuchasmicroorganisms/agents,plantcells
andanimalcells.Livingorganismsderivedfromsuchgeneticmodificationscarrydesired
phenotypesforuseinvarioussectorssuchaspublichealth,agriculture,industryand
theenvironment.
Overthepast40years,recombinantDNAtechnologyhasbeenextensively
exploited invarious industries suchas
thepharmaceuticalandmedical supply
industriesforhumanandanimaluses.Thisisexemplifiedbytheproductionofinsulin
for
treatmentofdiabetesbymicroorganisms/agentsgeneticallymodified
tobe
capableofproducinghumaninsulinandtheproductionofhumangrowthhormones
bygeneticallymodifiedmicroorganisms/agentstocuregrowthhormonedeficiency
inchildren.RecombinantDNAtechnologyalsoallowstheproductionofbiological
substances suchaspenicillin, vitaminB2andbioremediationagents
ingreater
amounts,athigherqualityandlowercosts.Additionally,recombinantDNAtechnology
has lead to thedevelopmentofdrugsandvaccines fordisease
treatmentand
preventionaswellasdiseasediagnostics,andmayenablethemedicalindustryto
producebiologicalsubstancesfortreatmentofcurrentlyuntreatablediseasessuch
ascancersandsomeinfectiousdiseases.Thefoodindustryalsobenefitsfromsuch
technology,asseenintheproductionoffoodandfood-relatedsubstancessuchas
enzymes,aminoacids,chemicalsandfoodadditives.Asfarastheagriculturalsector
isconcerned,desiredcharacteristicsofplantsandanimalscanbeaugmentedusing
thistechnology;geneticallymodifiedplantscanbegeneratedtocontrolinsectpests,
surviveindefinedenvironmentalconditionssuchasincoldordryweather,orfortified
toprovideimprovednutrition,whilegeneticengineeringofeconomicallyimportant
livestockmayenhancegrowthandimmunitytodiseases.
Inorder topromote theapplicationofGMMs for industrialuse
inboth
developedanddevelopingcountries,internationalbodiessuchastheOrganization
forEconomicCooperationandDevelopment (OECD)establishedguidelines
for
industrial applicationsofGMMs in1986, followedbya revision
in1992.These
guidelines,designedtoensurehumanandenvironmentalsafetyinconjunctionwith
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2 Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales
GMMuse,havebeenadoptedwithcertaindegreesofmodificationinanumberof
countriesbasedupontheunderlyingprinciplesthatmicroorganisms/agentshave
beenused in the foodandpharmaceutical industries fora
longtimeand that
associatedindustrialproceduresaresafegivenclearguidelinesonmicroorganism/
agentcontainmentaswellasspeciallydesignedequipmenttopreventtheirrelease
intotheenvironment.
Ingeneral,GMMsarenotradicallydifferentfromtheirparentalstrainsexcept
formodificationfordesiredcharacteristics.FollowingtheGoodIndustrialLargeScale
Practice (GILSP) thathasbeenmandatory
forassessinguseofunmodified
microorganisms/agentsinindustry,GMMswhichhavepassedthesafetyassessment
canbeeligibleforuseatindustrialscales.GMMscurrentlyusedinindustryaremostly
classifiedat theGILSPsafety level.OECDhassuggestedthatcountries
formulate
domesticguidelinesforassessingGMMindustrialapplicationbytakingintoaccount
harmfuleffectstheymaycausetohumansandtheenvironment,andhassupported
theapplicationofsafeGMMsatpilotandindustrialscales.IncaseswhereGMMs
areassessedtohavepotentialriskstohumansandtheenvironment,stringentsafety
controlsthroughtheimplementationofhighercontainmentlevelsandhighersafety
levelsforworkingproceduresarerequired.
For more information, please contact:
TheSecretariatoftheTechnicalBiosafetyCommittee
NationalCenterforGeneticEngineeringandBiotechnology(BIOTEC)
NationalScienceandTechnologyDevelopmentAgency(NSTDA)
113ThailandSciencePark,PhahonyothinRoad,KhlongNueng,
KhlongLuang,PathumThani12120,Thailand
Tel:+66(0)2-564-6700Fax:+66(0)2-564-6703e-mail:[email protected]
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Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 3
Chapter 2Scope and Principles
Theobjectiveoftheseguidelinesistoprovideguidanceforcontaineduseof
GMMsatpilotandindustrialscalestoensuresafetytooperators,thecommunity,
andtheenvironment.Thescopeandprinciplesoftheguidelinesareasfollows:
1.
Theseguidelinesfororganizationssuchasstateenterprises,privateand
governmentresearchinstitutes,industrialfactories,andprivatecompanies,where
GMMsarecultivatedorused tocommerciallyproducebiological
substances for
variousindustrieswithnointenttoreleaseGMMsintotheenvironment.
2.GMMs in
theseworkingguidelinesaremicroorganisms/agentswhose
genesorgeneticmaterialhavebeenmodified from itsoriginal
counterpart in
amannerthatcannotoccurinnaturethroughgeneticmodificationtechniquesfor
expressionofdesiredphenotypessuchasenzymeproduction.Theyincludeprogeny
ofsuchmicroorganisms,whichhaveinheritedthemodifiedgeneticmaterial.
3.
Geneticmodificationtechniquesreferredtointheseworkingguidelines
are:
3.1TheuseofrecombinantDNAtechnologytoligateDNAfragmentsor
heterologousgenesof
interestwithvectorsfollowedbytransformationintohost
cellsbymethodssuchaselectroporationtoenablesuchhostcellstoexhibitdesired
phenotypes.Plasmidsandvirusesareexamplesofvectorsused.
3.2IntroductionofDNAfragmentsorgenesofinterestintohostcellsvia
micro-injection,macro-injectionandmicro-encapsulation.
3.3Cellorprotoplast fusionandhybridization techniquesbetween
differentcelltypeswithdifferentgeneticmaterialstogenerateheterologousgenes
inmicroorganisms/agentsinamannerthatcannotoccurinnature.
4. AnyworkusingGMMsinpilotplantsandindustrymustundergosafety
assessmentstoensuresafetytooperators,thecommunityandtheenvironment.
TheseguidelinesclassifytheuseofGMMsatpilotandindustrialscalesinto4classes
accordingtotheworksafetylevelandlevelofriskfromGMMs.OnceGMMactivities
areclassified,appropriate containmentandbiosafety levels
canbeadoptedas
protectivemeasurestopreventreleaseorexposureofGMMstooperatorsandthe
environment.
5.Thesafetyor riskassessmentofGMMactivitiesatpilotand
industrial
scalesisbaseduponscientificinformationregardingGMMs,hostcells,vectors,genes
orDNAof interest,methodofgeneticmodificationandother factors
related to
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4 Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales
pathogenicity,allergy,andotherdiseasesinhumansaswellasnegativeimpacton
theenvironment.Therefore,
riskassessmentmustbeconductedbyabiosafety
committeeempoweredbyanorganizationorinstitution.
6.Theownerorauthorizedrepresentativeisrequiredtosubmitanapplication
forpermissiontouseGMMsatpilotandindustrialscalestotheauthorities(willbe
announcedlater),andapprovalmustbeobtainedpriortooperationcommencement.
Thisprocesscanbeinitiatedbytheownerorauthorizedrepresentativealongwith
thenewplantapprovalorpermitrenewalprocess.Moreinformationforapproval
processesisdecribedinChapter5.
7.Some techniquesmay result in some formofgeneticmodification
to
microorganisms/agents,butsomeofthesegenetically-alteredmicroorganisms/agents
aretechnicallynotconsideredGMMs,andthusarenotcoveredbytheseguidelines
(seelistofnon-GMMsinAppendix1).
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Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 5
Chapter 3Classification of GMM Work at Pilot and Industrial
Scales
GMMpracticesatpilotand industrial scalesareclassifiedaccording
to
thedegreeofsafetyandlevelofriskfromtheuseofGMMs.FollowingOECD,1992,
GMMworkisclassifiedinto4classesas:
GILSP
WorkusingGMMsclassifiedassafeandimplementinggoodindustrial
largescalepractice.
Class 1 WorkusingGMMsclassifiedas safebutdoesnot
fulfillGILSP
conditions.
Class 2 WorkusingGMMs thatmaypose low risks tooperators, the
communityortheenvironment.
Class 3
WorkusingGMMsthatmayposeriskstooperators,thecommunity
ortheenvironment.
3.1 GILSP
WorkinthiscategoryinvolvestheuseofGMMsthatdonotcauseanyharm
andadoptsgoodindustriallargescalepractice.GMMsusedmustbenon-pathogenic,
mustnotinvolveanyviralDNA,bacteriophageorplasmidthatmaycausedisease,
andmustbederivedfrommicroorganismsthathavealonghistoryofsafeusein
industryorhavelimitedsurvivalinthenaturalenvironment(Appendix2).GMMsin
thiscategoryarethoseclassifiedinRiskGroup1(Appendix4)orclass1inthebiosafety
guidelinesforlaboratorypractice.ExamplesincludeworkusingTBCsafety-approved
hostndvectorsystems(Appendix3)suchastheEscherichia
coliK-12,Saccharomyces
cerevisiae, Bacillus subtilis or Bacillus
licheniformishost-vectorsystems.
3.2 Class 1
WorkinthiscategoryinvolvestheuseofGMMsthatdonotcauseanyharm
butdonotfulfilltheGILSPconditionsabove.Itrequirestheminimumoflarge-scale
containmentlevel1(LS1).
Workinthisclass:
1.WorkusingGMMsclassifiedinRiskGroup1(Appendix4)thatdoesnot
fulfillGILSPconditions(Appendix2).
2.WorkusingGMMsclassifiedas class1 in thebiosafetyguidelines
for
laboratorypracticethatdoesnotfulfillGILSPconditions.
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6 Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales
3.3 Class 2
WorkinthiscategoryinvolvestheuseofGMMsthathavelowpotentialto
causeharmtooperators,thecommunityortheenvironment.Itrequiresaminimum
oflarge-scalecontainmentlevel2(LS2).
Workinthisclass:
1.WorkusingGMMsclassifiedinRiskGroup2(Appendix4).
2.
WorkusingGMMsfromsafety-approvedhost/vectorsystems(Appendix3)
whichcontainDNAorgenesofinterestthat:
- maycauseorbe involved in thedevelopmentofdiseases, cancer,
toxicity,adverseeffectsongrowthorcelldivision,orotherpathological
effectsonhumans,animalsorplants;or
- areuncharacterizedDNA/geneswithunclearfunction.
3.4 Class 3
WorkinthiscategoryinvolvestheuseofGMMsthatarepotentiallyharmful
tohumanhealth,thecommunityortheenvironment.GMMsthatfallintothisclass
maycausediseasebutnotdiseaseepidemics,andsuchdiseasescanbeprevented
andtreated.Thisclassalsoincludesworkwithanunidentifiedlevelofrisk.Itrequires
aminimumoflarge-scalecontainmentlevel3(LS3).
Workinthisclass:
1.Workusingtoxin-producingGMMs,includingGMMswithDNAthatcontrol
toxinproductionorproducetoxinspossessinganLD50oflessthan100ng/kg(Appendix5),
orworkinvolvinggenesproducingtoxinswithanLD50lessthan100ng/kg,orwork
involvingDNAfromGMMsthatproduceunidentifiedtoxins.
2.WorkusingGMMsthatincludeviralvectorswhichcaninfecthumancells,
andwork involvingmodifiedDNAwith theability
toproducegrowth-controlling
substancesortoxicsubstancestohumancells.
3.WorkusingGMMsthatincludevectorsorhostsfrommicroorganisms/
agentsinRiskGroup3,whichhavepotentialtocausediseaseinhumansorcertain
diseases in plants or animals.
4.WorkusingGMMsthatincludewholeviralgenomesorviroids,orgenetic
materialswhichcaninfecthumans,animalsorplants.
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Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 7
5.WorkusingGMMsinvolvingligationbetweenwholeviralgenomes,viroids
andcomplementary fragments that cancause infectionorare
important to
thedevelopmentofdisease.Italsoincludesworkinvolvinginfectionofhostcellsor
increasingmicrobialvirulenceorinfectivity.
6.WorkusingGMMswithmultipleantimicrobialresistancegenes,where
thoseantibioticsarestillusedfortreatmentofinfectiousdiseasesinhumans,animals
orinagriculture.Theseantibioticresistancegenesmustbeidentifiedastowhether
theycanbenaturallytransferredtoothermicroorganisms/agentsornot.
Remarks: 1)
Geneticallymodifiedmicroorganisms/agentsacceptedassafeareclassifiedasGILSP.
2)Safehosts/vectorsexpressingvirulencegeneswillbeconsideredonacase-by-casebasis.
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8 Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales
Table 3.1:SummaryofGMMworkatpilotandindustrialscales
Class Risk Group* Description Containment Examples
GILSP 1 UseofGMMsthathave GILSP Bacteria
beenclassifiedassafe - Bacillus subtilis
- Bacillus megaterium
- Streptococcus thermophilus
Yeast
- Saccharomyces cerevisiae**
- Schizosaccharomyces pombe
Class1 1 UseofGMMsthathave LS1 Bacteria
beenclassifiedassafe - Bacillus licheniformis
butnotfulfillingGILSP non-sporeforming
conditions Virus
- Adeno-AssociatedVirus
(AAV)Types1-4
Class2 2 UseofGMMsthatmay LS2 Bacteria
poselowrisksto - Clostridium botulinum
operators,community - Corynebacterium diphtheriae
ortheenvironment - Staphylococcus aureus
- Vibrio cholerae
Class3 3 UseofGMMsthatmay LS3 Bacteria
poseriskstooperators, - Mycobacterium tuberculosis
communityorthe - Yersinia pestis
environment,andmay Rickettsia
causediseasethatcan - Rickettsia akari
bepreventedand
treated and do not
causeepidemics
*
RiskGroupofmicroorganisms/agentsaccordingtoNIHGuidelinesforResearchInvolvingRecombinantorSyntheticNucleicAcid
Molecules(2013).
**UseofSaccharomyces
cerevisiaesubtypeboulardiiisprohibitedasitpresentsadangertosusceptiblepeople,includingpatients
withcentralvenouscatheters.
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Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 9
Chapter 4GMM Containment Levels for Pilot and Industrial
Uses
ContainmentisdefinedasthecontrolofGMMsinarestrictedfacilitywith
theaimofpreventingtheirspread
intotheexternalenvironment.Therearetwo
typesofcontainment:biologicalcontainmentandphysicalcontainment.Biological
containment preventsGMMs fromsurvivingor transferring
itsgeneticmaterials
outsideabioreactor,whereasphysical
containmentrequiresasuitabledesignand
installationoffacilities,equipmentandworkingareas,aswellasaworkingprotocol
topreventthereleaseofGMMsintotheenvironment.Safetyofpilotandindustrial
applicationsofGMMscanbeachievedbyimplementingappropriatecontainment
measures.
Intheseguidelines,fourcontainmentlevelsareidentifiedaccordingtodegree
ofsafetyandriskassociatedwiththeGMMsandothercriteriasuchastheamount
ofGMMintheproductionprocessandthepurificationprocess,whichmayalterthe
levelofcontainment.Acombinationofcontainmentlevelscanbeadoptedwithina
singleworkingenvironmentdependingonsafetyassessmentresults.Forinstance,
ina facilityoperatingat containment level1, it is alsopossible
to incorporate
containmentlevel2measuresforallorspecificpartsoftheworkaccordingtoother
safetyconsiderationstoensurethesafetyofoperatorsorpersonnel.Thecontrolof
GMMbiosafetyorcontainmentinindustryislargelyidenticaltothatimplemented
inlaboratories,althoughmorestringentpracticesandahigherdegreeofcautionare
requiredsincetheworkingvolumeofGMMsatpilotandindustrialscalesishigher,
andadverseeffectsonhumansandtheenvironmentwouldaccordinglybemoresevere.
The regulationsapplied toall classesusingGMMsare listedas
follows
(alsoseeAppendix6):
1.Workingproceduresmustbeclearlydescribed.
2.EquipmentandtoolsusedforGMMworkmustberegularly inspected,
accordingtoGMMclassification.
3.InspectionofcontaminationorreleaseofGMMsbothinthecontained
workingareasorthesurroundingenvironmentisrequired.
4.Inactivation/eradicationofGMMsandculturefluidbeforebeingreleased
intotheenvironmentmustbedoneusingappropriatemethods.
5.Emergencyplansmustbefollowedincaseofextensivespillageorrelease
ofGMMs.
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10 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
6.Trainingmustbeprovided foroperatorsorpeople involved for
anunderstandingofworkandsafetypractices,andproperemergencydrillsmustbe
conductedregularly.
7.AnIBCmustbeestablishedtocoordinateGMMwork.
Containmentintheseguidelinesisdividedintofourlevels(seeAppendix7),
asfollows:
4.1 Good Industrial large Scale Practice (GILSP) Containment
GILSPcontainmentreferstocontainmentapplicabletoGILSPworkatpilot
andindustrialscales.Thiscontainmentexercisesthelowestlevelofbiosafetycontrol
conformingtothegeneralpracticesoutlinedaboveandinAppendix6.GILSPGMMs
areconsideredsafeandthereforearenotsubjecttocontainmentinaclosedfacility.
However,precautionsmustbetakentopreventdirectcontactwithGMMsorspillage.
Sampling,inoculationortransportfromonesystemtoanothermustbeconducted
withcaretopreventcontaminationorexposuretooperators.GMMsandculture
fluidmustbeinactivatedbeforebeingdischargedfromthesystem.Healthsurveillance
isnotrequiredforthisclassofcontainment.
4.2 Large-scale Containment Level 1 (LS1)
Large-scale containment level1 refers to
thecontainmentapplicable to
class1GMMwork.ThiscontainmentlevelfollowsthegeneralpracticesinAppendix6
andadditionalrequirementsasfollows:
1.Facilitylayout,workingareaandworkingprotocolsmustbewellplanned.
2.GMMsmustbecontained ina closed system (suchasa reactor)or
appropriatecontainmentequipment(suchasabiosafetycabinet).ReleasedGMMs,
ifany,mustbesominimalthattheydonotcauseharmtooperators.
3.TransportofGMMsduringworkingprocedures,includingsamplingand
inoculation,mustbecarriedoutwithconsiderablecaution,andaerosolsreleased
duringtransportmustbeminimized.
4.Reactorsorequipmentmustbedesignedtominimizeexhaustgas/aerosols.
Exhaustgasfromaclosedfacilitymustbeemittedthroughahighqualityfilterofat
leastHEPAstandard.Equallyeffectivemethods forGMMneutralization,
suchas
incinerationorchemicaltreatment,canbeusedtominimizereleaseofGMMs.
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 11
5.Afteroperation,reactorsorequipmentmustbesterilizedbeforebeing
opened,washedornextuse.Routinevalidationofsterilizationprocessisrequired.
6.
Incidentsofspillageorcontaminationmustbereportedtothebiosafety
officerandotherresponsiblepersons,includingtheownerorauthorizedrepresentative
(suchasprojectorinstitutiondirectors).Medicaltreatmentaswellascasefollow-ups
andtherapydetailsofpatientsaffectedbyspillageorcontaminationmustberecorded.
7.Operators’healthsurveillancemustbeimplemented.
8.Emergencyplansmustbefollowedincaseofextensivespillageorrelease
ofGMMs.
9.GMMs inwastemustbeneutralizedbeforebeing released into the
environment.
4.3 Large-scale Containment Level 2 (LS2)
Large-scalecontainmentlevel2referstocontainmentapplicabletoclass2.
ThiscontainmentlevelfollowsthegeneralpracticesinAppendix6andlarge-scale
containmentlevel1practices,withadditionalrequirementsasfollows:
1.EquipmentindirectcontactwithGMMsmustbespeciallydesignedto
allowsterilizationbyheatorchemicals,inactivatingGMMsbeforeopeningorcleaning.
2.Equipmentsuchas rotatingsealsorothermechanicaldevicesused
in
GMMcultureprocessesinaclosedsystemmustbeproperlysealedtopreventrelease,
orplacedwhereexhaustgascanbereleasedthroughahighqualityfilterofatleast
HEPAstandard.Equallyeffectiveneutralizationmethodscanbealsoimplemented
andmustberoutinelytested.
3.Bioreactorsandotherequipmentintheclosedsystemmustbeequipped
with sensors to monitor containment.
4.Closuremonitoringofclosedsystemsmustbeimplementedtoensureno
releaseofGMMs.
5.Closureintegritymustbevalidatedagainsthostorganisms.
6.ClosedsystemequipmentforusewithGMMsmustbeusedforthispurpose
only.Recordsmustbekeptforalluseofsuchequipment,includinguseinresearch,
systemtestingandproduction,aswellastheirmaintenance.
7.Ventilationingasexhaustareasmustbemaintainedusinghighquality
filtersofat
leastHEPAstandardoranequivalentprocessandmustbetestedon
aregularbasis.
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12 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
8. Onlypertinentoperatorsmayhaveaccesstocontainedareas.
9.Safetyplansandemergency trainingmustbeprovided
forpertinent
operators so that theymanageemergency situations
suchasGMMspillageor
contamination.Emergencyprotocolsmustbepostedinworkingareas.
10.Emergencyequipmentandtoolsmustbelocatedinworkingareasand
routinelycheckedtoensurethattheyareingoodconditionatalltimes.
11.Signsdisplayingcontainmentlevelsmustbepostedinthecontainedareas
andonequipmentforGMMuse.IncidenceofspillageorreleaseofGMMsmustbe
reportedtotheIBCandTBCimmediately.
4.4 Large-scale Containment Level 3 (LS3)
Large-scalecontainmentlevel3referstocontainmentapplicabletoclass3.
ThiscontainmentlevelfollowsthegeneralpracticesinAppendix6andlarge-scale
containmentlevel1andlarge-scalecontainmentlevel2practices,withadditional
requirementsasfollows:
1.AnyworkrelatedtotheuseofGMMsinculturemediamustbeperformed
inaclosedsystemorinapprorpiatecontainmentequipment(suchasalevel3biosafety
cabinet).Activitiesthatinvolvetheuseoflessthan10litersofGMMscanbeconducted
outside theclosed systembutmustbemaintainedwithinphysical
containment
conditionsidentifiedinAppendixG-II-CofbytheNIHguidelines(2013).
2.GMMsmustnotbereleasedfromclosedsystemsorbasiccontainment
equipmentunless the
sterilizationprocesshasbeenvalidated.Validationof
sterilizationherereferstovalidationofthesterilizationefficacyofhostorrecipient
cells.CulturemediacontainingtheendproductsofGMMsorviralvectorsmaybe
removed fromtheclosed systemorbasic containmentequipment,whether
for
laboratoryanalysis,useinotherprocessesorforpackaging,onlybyemployingclosed
systemtechniques.
3.Closed systems forpropagatingGMMsmustbe speciallydesigned
to
preventoverflowofculturemediumduringcultivation.
4.Containedareasmustbedesignedtohavegoodcontrolofaircirculation,
allowingairtoflowfromlesscontaminatedtomorecontaminatedareas.Systems
shouldbedevelopedtopreventreverseairflowandalarmsshouldactivateifreverse
airflowoccurs.Airfromrestrictedareasmustnotbeusedinotherworkingareas.
Exhaustgas/air shallpass
throughaHEPAfilteroranequivalentfiltrationor
inactivationmethodpriortodischargefromthesysteminordertoremoveGMMs.
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 13
5.Restrictedareasmustbeaccessed throughseparateentrancesand
beequippedwithdouble-dooredspacessuchasairlocksorpartitionsseparating
therestrictedareasfromotherareas.
6.Restrictedareasmustbesealedforhigh-efficiencyGMMdecontamination
byfumigationorotherdecontaminationmethods.
7.
RestrictedareasmustbedesignedtopreventreleaseofGMMsintoareas
outsidetheclosedsystemincaseofGMMspillageorleakagefromcontainedareas
orbasiccontainmentequipment.
8.Change roomsequippedwith showersmustbeprovided in
restricted
areasforusebyoperators.*
9.Operatorsmustwearlaboratorygowns,putonshoeorfootcovers,and
showerbeforeenteringandleavingrestrictedfacilities.
10.Handwashingisrequiredbeforeexitingrestrictedareasusinghandwashing
appliancescontrolledbyelbowsorfeet,oranyotherkindofautomatic,no-touch
handwashingequipment.*
11.Useduniformsshallbewashedproperlyordestroyed.
12.Personsunder18yearsoldarestrictlyprohibitedfromenteringrestricted
areas.
13.Infrastructuresystemsincludingmaintenance,sewers,wiring,telephone
linesoranyothercommunicationsystemsmustbeinstalledusingspeciallydesigned
materialstopreventcontaminationofGMMs.
*Effluentsfromhandwashingsinksandshowersandothercontaminatedeffluentsmustbeinactivatedaccording
toriskassessmentbeforedischarge.
-
14 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 15
Chapter 5Approval Process for Projects with Contained Use of
GMMs
at Pilot and Industrial Scales
TheapprovalprocessfortheuseofGMMscanbeinitiatedbytheowneror
authorizedrepresentativewiththeauthorizedorganization(willbeannouncedlater)
alongwith thenewplantapprovalorpermit renewalprocess.Documents
for
considerationareasfollows:
- Scientificnameofmicroorganism/agent
- Sourceofmicroorganism/agent
- TechniquesusedforGMMdevelopment
- Historyofuse
- Purpose(s)ofuse
-
ContainmentandsafetymeasuresfortheuseofGMMsatindustrialscales
- EmergencyproceduresforspillageorreleaseofGMMs
- Certifieddocumentsofresponsibleperson
- Evidenceofbiosafetytraining(ifany)
-
ImportpermitorlicensefromrelatedagenciessuchastheDepartment
ofMedicalSciencesortheDepartmentofAgriculture(ifany)
FortheuseofGMMs,theownerorauthorizedrepresentativemustfollow
theBiosafetyGuidelinesforContainedUseofGeneticallyModifiedMicroorganisms
atPilotandIndustrialScales.Additionally,theownerorauthorizedrepresentative
musthold safety certificates to confirm the
safeuseofmicroorganisms/agents.
Fortheuseofclass2or3GMMs,theresponsibleauthoritywillbeannouncedlater
andpermissionforusemustbegrantedcasebycase.
-
16 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
Figure 5.1
TheapprovalprocessflowchartforprojectswithcontaineduseofGMMs
atpilotandindustrialscales
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 17
Chapter 6Risk Assessment for Contained Use of GMMs at Pilot
and
Industrial Scales
CarefulandthoroughriskassessmentforcontaineduseofGMMsatpilot
andindustrialscalesmustbeconductedwithgreatconsiderationforthepotential
risksposedtohumansandtheenvironmentfromGMMuse,workingprocedures
andtheamountofGMMs.
6.1 Criteria for risk assessment
1. GMM risk group
Microorganisms/agentsareclassifiedintofourriskgroupsaccordingto
theirrelativepathogenicitytohumans(Appendix4)aslistedbelow:
-
Riskgroup1consistsofmicroorganisms/agentsthatarenotassociated
withdiseasesinhealthyadults.
-
Riskgroup2consistsofmicroorganisms/agentsassociatedwithdiseases
thatarerarelyseriousandgenerallycontrollablethroughtreatment
andpreventionmeasures.
- Riskgroup3consistsofmicroorganisms/agentsthatareassociated
withserioushumandiseasesbutarecontrollablethroughtreatmentand
preventionmeasures(highrisktoindividualbutlowrisktothecommunity).
-
Riskgroup4consistsofmicroorganisms/agentsthatcauseseriousand
fataldiseaseswithnotreatmentandpreventionmeasures(highrisk
tobothindividualsandthecommunity).
2. Risk of harm to humans and the environment
Riskmustbeassessedfromthetypeofhostcell,vectororinsertedDNA
used;thetechniquesusedfortheirmodification;microorganism/agentpathogenicity,
virulence,transmissionanddegreeofsurvivalinthenaturalenvironment;working
procedures;andtheamountofGMMsused.
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18 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
Afterriskassessment,theappropriatecategoryofworkandcontainment
leveslshallbeselectedforimplementation.Thecontainmentlevelrequiredmaybe
equivalenttotheriskgroupclassificationoftheagentused,oritmaybehigheror
lowerasa resultof theaboveassessment. For instance,DNAorgenes
from
microorganisms/agents in
riskgroup1,whicharegenerallynon-pathogenic,
mayexpresstoxicproducts,exhibitincreasedpathogenicity,orinduceallergicreactions
whenintroducedintohost,thereforenecessitatingahigherlevelofcontainment.
6.2 Required scientific information for risk assessment
The required scientific information for riskassessmentofGMMwork
is
summarizedbelow(forclass2GMMsorhigherseedetailsinAppendix9):
1.GMMinformation
-
Informationregardinghostconsistsoftheircommonname,scientific
andstrainnameincludingclassificationlevel,taxonomy,historyofprior
geneticmodification,pathogenicity,survivabilityinenvironment.
- Information regardingvectorand insertedDNAorgeneconsistsof
characteristicsandhistory,preparationandligationmethod;stability
in host cells and mobilisability.
- InformationregardingGMMsconsistsofexpressionofinsertedDNA
orgene,comparisonofcharacteristicswithhostorrecipientcelland
survivabilityinenvironment
2.
InformationregardingGMMworkrequiresconsiderationoftheriskto
humansand theenvironment,which isbasedonGMMpropagation
conditions,theamountsofGMMsused,anddownstreamprocessingand
purification.
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 19
Chapter 7Safety Management System for Contained Use of
GMMs at Pilot and Industrial Scales
ToensurethesafetyofworkinvolvingcontaineduseofGMMsatpilotand
industrialscales,itisnecessarytospecifytherolesandresponsibilitiesofeveryone
involved inGMMwork in theorganization,as set forth in
theguidelines.Their
responsibilitiesincludeperformingsafetyassessments,specifyingappropriateclass
andcontainment levelsaswell aspreventionmeasures,and
reportingpossible
problems thatmayaffectanyaspectof safety.Thebiosafetyofficerand
specific
persons in charge, suchas themanufacturingmanager,
shouldbedesignated to
manageworksafetyandanInstituteBiosafetyCommittee(IBC)shouldbesetupto
conductworksafetyassessments.
ManagementofworksafetyregardingcontaineduseofGMMsatpilotand
industrialscalesisdescribedbelow.
7.1 Roles and responsibilities of various personnel in the
organization/institution
1. Director/Head of the GMM operation unit (manufacturing
manager)
ThispersonmustclearlyunderstandtheBiosafetyGuidelinesforContained
UseofGMMsatPilotandIndustrialScalesandundertakethefollowingresponsibilities:
-
Coordinatingwithoperatorstofacilitatetheimplementationofcontrol
measuresforthesafestworkingconditions,accordingtotheguidelines.
- Arrangingforbiosafetytrainingprograms.
- Providingdetails
regardingGMMs,workclassification,andworking
proceduresduringinspections.
-
Ensuresthattheoperatorsadheretoregulationsregardingaccessto
the restricted areas.
-
SettingupasystemforrecordingdetailsconcerningGMMwork,suchas
1)NameofGMMsbeingused,
2)PurposeforusingGMMs,
3)AnalysisofGMMproperties,alongwithdate,timeandsampling
location,
4)StorageandtransferofGMMs.
- Arrangingforannualhealthinspectionsforoperators.
- CollaboratingwiththeIBCtoreviewGMMsafetymeasures.
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20 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
- Settingupa system for recordingdetails concerning
inspectionof
equipmentdirectlyexposedtoGMMsandotherequipment,including
sensing tools.
-
Organizingtrainingprogramsonthehandlingofemergencysituations
forallpersonnel.Thisprogramshall include information
regarding
emergencyproceduresandequipment,chemicalsandproceduresfor
emergencyreportingtorelevantsupervisors.
2. Institutional Biosafety Committee (IBC)
TheIBCshouldcomprisebothtechnicalandacademicexpertsinvarious
fieldstomakedecisionsregardingGMMwork.Examplesofrecommendedexperts
include:
- Personnelwith theknowledgeand skill toassessandexamine
thesafetyofGMMworkforoperatorsandtheenvironment.
- Biosafetyofficer(ifany).
- Experiencedengineertoexaminethesafetyofbiologicalequipment
topreventdisseminationofGMMs.
- Owner,directororauthorizedrepresentatives
- Externalexpertswithknowledge,expertiseandcapacitytoprovide
suggestionsonsafeGMMuse.
ResponsibilitiesoftheIBC
- AssessingGMMrisks,classifyingworkandGMMlevels,andproviding
suggestionsconcerningcontainmentsystemsandsafetymeasuresto
ensurecompliancewithbiosafetyguidelinespriortocommencementofwork.
-
Providingadviceandsuggestsregardingworksafetyinmatterssuchas
1)WorkingproceduresforGMMwork,
2)Trainingandhealthsurveillance,
3)Improvingworkproceduresandotheressentialsafetyconsiderations
inordertominimiseorpreventaccidents,and
4)Otherfactorsnecessaryforworksafety.
- Reviewing reportsand relatedproceduresperiodicallyorwhen
necessary.
- PreparingemergencyplanforspillageorreleaseofGMMs.
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 21
3. Biosafety officer
Biosafetyofficersshouldbeexpertsoncontrolofandprotectionagainst
biohazards,beknowledgeableconcerningtheBiosafetyGuidelinesforContained
UseofGeneticallyModifiedGMMsatPilotandIndustrialScalesand/orequivalent
Biosafetyguidelines,beabletoprovideadviceregardingsafetyissues,andorganize
safetytrainingforoperatorsandnewpersonnel.Theymustalsoensurethatwork
proceduresineachstepfollowtheworkingguidelines.Theofficerswillliaisewith
theIBCandproviderelevant
information.Substituteofficersmustbeassignedin
casethemainofficersareabsent.
4. Operator
Operatorsshouldhaveclearknowledgeandunderstandingaboutsafety
issues,carefullyimplementproperworkingproceduresforoccupationalsafety,and
shouldabletogivesafetyadvicetothosewhoarenotdirectlyinvolvedinGMMwork
butneedtoorareallowedtoaccesstheworkingareas.
7.2 Training for operators
Trainingprogram(s) shallbeorganized forall relatedoperators
regarding
workingprocedurespriortoactualoperation.Thefollowingtrainingtopicsarehighly
recommended:
1.KnowledgeandunderstandingofsafetyissuesassociatedwithGMMsuse.
2.ClassificationofGMMworkaccordingtorisklevels.
3.Know-howregarding techniquesanddevicesused toensure safety
to
operatorsandpreventthedisseminationofGMMs.
4.Significanceofworkingproceduresdesigned to improve safety
for
operatorsandtheenvironment.
5.Workingproceduresunderemergencysituations.
7.3 Health surveillance of operators
Theowner/directoroftheorganizationshallberesponsibleformonitoring
operatorhealth,asfollows:
1.PhysicalexaminationsfornewoperatorsbeforestartingGMMworkand
foralloperatorsannually.
2.
InthecaseofGMMworkclassifiedasclass2or3,preventionmeasures
tomaximizesafetymustbeimplementedpriortooperationandspecifictreatments
fordiseasescausedbyGMMsusedmustbepreparedandavailable.
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22 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
3.
InthecaseofexposuretoGMMsclassifiedasclass2or3,intensivemedical
check-upsbyqualifiedphysiciansaswellasbloodtestsandfollow-upsonsymptoms
oreffectsofdiseasesmustbeconducted.
4. Inthecaseofworkwithclass3GMMs,operatorbloodsamplesmustbe
drawnpriortocommencingGMMworkandkeptforatleast10yearsaftercompletion
oftheworktoallowmonitoringforcausesofsicknessordiseasethatmaysubsequently
develop.
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 23
Chapter 8Waste Management of GMMs
Accordingtointernationalguidelines,allcontaminatedliquidorsolidwaste
mustbedecontaminated/inactivatedbyvalidatedmeansbeforedisposal.Thetreated
wasteshallnotcontainany transferrablegene/DNAtoensure that
itwillnotbe
disseminatedintotheenvironment.
For work classified as GILSP or class
1,inactivationofcontaminatedmaterials
andwasteisrequiredbyusingvalidatedmeans.FormaterialscontainingGMMsthat
undergooff-siteinactivation,theregisteredwastecontractorhiredtoremovethe
wastemustholdpermitNo.101forfactoryoperationissuedbytheDepartmentof
IndustrialWorks.Moreover,thedetailsofhowwasteistreatedanddisposedbythe
contractormustberecorded.
For work classified as class 2 or class 3, contaminated
materials and waste
mustbeinactivatedatthesitewherecontaineduseactivitytookplace.However,
viableGMMcellsfromclass3workmustbeinactivatedby heat
sterilization on-site
(i.e.materialsmustnotbe removed fromcontainment for
inactivation).Exhaust
gasesfromclass2and3closedsystemsmustalsobetreatedtopreventtherelease
of viableorganisms.Additionally, inactivationof class3GMMs
ineffluents from
hand-washingsinksandshowersorsimilareffluentsisrequired.
8.1 Inactivation method(s)
Inactivationmethod(s)chosenmustbeappropriatetotheGMMriskgroup
andinactivationefficacymustbevalidatedagainsttheorganismbeingused.
Large-scale effluents can be treated by chemical or thermal
methods or
acombinationofboth,andpossiblycombinedwithpressure.Heatinactivationis
generallyconsideredmoreappropriateforlarge-scaledischarge,withacombination
ofheatandpressureneeded toensure thatallbiological
agentsaredestroyed.
Thecombinationofheatandchemicalshasanadvantageinthatinactivationrequires
nopressureandlowertemperaturescomparedtoasystembasedonheatalone.
However,appropriatetemperatureandchemicalcombinationsneedtobedetermined
forinactivatingtheagentsusedinthefacility.Also,chemicallyinactivatedeffluents
are still required to complywithphysicalandchemicalparameters
setdownby
wastewater regulations (Notificationof theMinistryof
IndustryNo.2,B.E.2539,
issuedundertheFactoryActB.E.2535,Re:IndustrialEffluentGuidelinesforFactory
Discharge)beforereleaseordisposal.
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24 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
Solid
wastescanbetreatedbyautoclavingfollowingthecycleparameters
(temperature,timeandpressure)inAppendix10.Anapprovedincinerationsystem
canbeusedasanalternativeapproach.
8.2 Verification and validation of decontamination/inactivation
methods
Thechosenmethodofinactivation(e.g.heatorchemicalinactivation)mustbe
verifiedandvalidatedunderworkingconditionstoensureitseffectiveness.
For
verification,theappropriatebiologicalindicators(Table8.1)mustbeused
periodicallyasacontrol.Thewastetreatmentmethodmustbetestedregularlyfor
efficiencyofdecontaminationandarecordofthetestresultsmustbekeptfor5
yearsforinspectionuponrequirement(RegulationoftheOfficeofthePrimeMinister
onRecordKeepingB.E.2526,Chapter3:Documentstorage,lendinganddestruction).
For
validation,theworst-casescenariomustbeperformedusingthehostcell
orequivalent.Conditionstobevalidatedincludetemperatureofheatinactivation,
concentrationofchemicalagentsorcontact/holdingtime,densityandvolumeof
GMMwaste,foreachtargetcelltype.
Frequencyofvalidationdependsonriskassessment(atleastonceayear,
undernormalworkingconditions).Recordsofvalidationsuchasvalidationprotocol
andtheresultsofthevalidationexercisemustberetainedbytheuserandkeptfor
5yearsforinspectionbyregulatoryauthoritiesuponrequest.
Table 8.1: Examplesofbiological indicators
forverificationofheatandchemical
inactivation.
Biological indicators
Heat Chemical Reference
inactivation inactivation
1. Bacillus atrophaeus* P P *Fleming/Hunt
(Chlorinedioxidegas, ASMbook,
Formaldehydegas) 3rdEd.&BMBL2007
2. Bacillus coagulans** P Î **SporeNews,
3. Bacillus subtilis var. niger* P Î biological indicators
4. Clostridium sporogenes** P Î newsletter,Volume
10No.1.
5. Geobacillus (Bacillus) P P
stearothermophilus** (Hydrogenperoxide
vapour)
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 25
8.3 Waste (inactivated/non-inactivated) storage and
transport
GMMwastemustbecollectedandstoredinsecure,closed,andleakproof
containers(triplepackaged)withstatuslabelsandbiohazardsigns.Inthecasethat
thecontainersmustbekeptpriortodecontamination,accesstothestoragearea
mustberestrictedtoauthorizedpersonnelonly.
GMMwastetobetransportedmustbecontainedintriplepackagedcontainers
labeledwithbiohazardsigns,wheretheprimaryandsecondarycontainersmustbe
asecure,closed,andleakproof.
GMMs waste transport:
Ifwastemustbe transported, specialpractices shouldbedeveloped
for
transportofinfectiousmaterialstodesignatedalternatelocation(s)withinthefacility
(Notificationof theMinistryof
IndustryonLandTransportationofHazardous
SubstancesB.E.2546,andNotificationoftheMinistryofIndustryonIndustrialWaste
DisposalB.E.2548).TransportationofuntreatedwasteofGMMclass3isprohibited.
8.4 Waste disposal
Beforefinaldisposal,thepresenceofviablecellsinwastesamplesshallbe
monitoredbyculturinginenrichedmedium.Negativecontrolsshouldbeusedto
ensurethatanygrowthobservedisverifiablyderivedfromGMMsinliquidwaste
ratherthanexperimentalerror.Forworkinclass2andclass3,theabsenceofnaked
DNA inwastedisposalmustbeperiodically validatedby transformation
into
theoriginalhost (in thecaseofGMMsharboring
replicativeplasmids)orPCR
(GMMswithchromosomallyintegratedgenes).
WhereDNAisusedforpreventiveortherapeuticmedicine,non-functionality
ofDNAinwastedisposalmustbeassured.Thiscanbeachievedeitherbyreducing
DNAfragmentstonon-functionallengthsoralteringthestructureoftheDNA.
Moreover,GMMwastemanagement shall complywith theFactoryAct
B.E.2535(C.E.1992),thePathogensandAnimalToxinsActB.E.2525(C.E.1982),
thePathogensandAnimalToxinsAct(No.2)B.E.2544(C.E.2001)andcurrentlythis
acthasenforced.Companiesthatprovidewastehandlinganddisposalservicesmust
beauthorizedundertheHazardousSubstancesAct,B.E.2535,2544and2551.
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26 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
8.5 Waste records and labeling
RecordsofGMMinactivationevents(run-timeparametersandtestresults)
fortheprevious5years(RegulationoftheOfficeofthePrimeMinisteronRecords
KeepingB.E.2526,Chapter3:Documentstorage,lendinganddestruction)period
mustberetainedbytheuser,forinspectionbyauthoritiesuponrequest.Statuslabels
ontheoutsideofGMMwastecontainersmustprovidethefollowinginformation:
- Typeofwaste:solid,liquidorsharps
- Amountofwaste
- Scientificname,GMMriskgroupandclassofwork
- Name(s)ofcollector(s)anddate
- Nameofpersonresponsibleforwasteinactivation
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 27
Table 8.2:Wastemanagementrequirements
•
•
•
•
-
28 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 29
Chapter 9Emergency Plan and Inactivation of Spilled GMMs
in Contained Use at Pilot and Industrial Scales
OrganizationsorinstitutionsthatuseGMMsatpilotandindustrialscalesare
requiredtoprepareemergencyplansandmethodsofGMMsinactivationincaseof
spillageasdetailedbelow:
1.Emergencyplan(s)forresponsetoaccidentsmustbedesignedinorder
toensuresafetytooperatorsandtheenvironmentandthoseplansmustbeapproved
bytheIBCpriortoactualoperation.
2.Emergencyplan(s) shall
includecountermeasures,standardoperating
procedures (SOP)andnecessaryequipmentandchemicals.Periodic
reviewof
theemergencyproceduresandvalidationofequipmentarerecommended.
3.Emergencyincidentsmustbereportedtorelevantagenciesorunitsand
regulatoryauthorities(seeAppendix11forsampleincidentreportform).
4. Incidentreportsshallinclude
- Nameofthereporter
- Place
- Situation
- NameofGMM,includingitscharacteristicsandtheamountspilled,
and
- Othernecessary information forassessing thedangerposed to
operators,thecommunityandtheenvironment
5.
Inthecaseofextensivespillage,clearlyspecifythemethodsandprocedures
forGMMinactivation.Forinstance,drainsmaybefittedaroundbioreactorsinorder
toaccumulateleakedfluidsinacontrolledareaforchemicalorheatinactivation.
6.Emergencyincidentsshallbereportedannuallytoregulatoryauthorities
andIBCshouldkeepthereport(s)foratleast5years.
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30 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 31
Chapter 10Possession, Transport, Import and Export of GMMs
Possession,transport,importandexportofGMMsdiscussedinthischapter
appliestothetransferofGMMsinclasses1–3only.
Forclass1orclass2GMMs,thecontainershallbetight,closed,unbreakable,
abletoresistpressureandshocks,anddesignedtopreventcontentrelease.
Forclass3GMMs,boththeinnerandtheoutercontainershallbeimpermeable
to liquids.A
liquid-absorbingmaterialcapableofabsorbingtheentirevolumeof
transportedliquidshallbeplacedbetweentheinnerandtheoutercontainer.Ifmore
thanoneinnercontainerisplacedinthesameoutercontainer,eachinnercontainer
shallbewrappedinmaterialthatcanabsorbshocksandliquids.Theoutercontainer
shallbetight,closed,unbreakable,abletoabsorbpressureandshocks,andmust
preventcontentrelease.
10.1 Packaging and Transfer or Transport of GMMs
1.TheprimaryreceptaclecontainingGMMsmustbewatertight,leakproof
andappropriatelylabeledastoitscontents.Thisprimaryreceptacleiswrappedin
enoughadsorbentmaterialtoabsorballfluidincaseofbreakageorleakage.
2.Asecondwatertight,leakproofpackagingisusedtoencloseandprotect
theprimaryreceptacle(s).Severalwrappedprimaryreceptaclesmaybeplacedin
asinglesecondarypackaging.Volumeand/orweightlimitsforpackagedinfectious
substancesareincludedincertainregulatorytexts.
3.Athirdlayerprotectsthesecondarypackagingfromphysicaldamagewhile
intransit.Specimendataforms,lettersandothertypesofinformationthatidentify
ordescribe theGMMsand identify the shipperand
receiver,andanyother
documentationrequiredmustalsobeprovidedaccordingtolatestregulations.
4.
Inthecaseoftransportbyparcelpost,theouterpackageshallbemade
ofabsorbentmaterialsuchasthickpaperorwood,ormaterialwhichisnoteasily
broken. Labels shalldisplay commonandscientificnamesofGMMs
inEnglish,
quantity,day/month/yearofproduction,andproductionplace,andmustbear
avisiblewarningof“Danger”togetherwithcontactdetailsofthesendersforimmediate
contactincaseoflossordestructionduringtransfer.(SeeFigures10.1–10.3)
5.
InthecaseofliquidGMMtransport,biologicalspillkit(s)andabsorbent(s)
shallbesufficientlyavailableformanagementofspills.
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32 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
10.2 Possession, Import and Export
1.Possession,importorexportofGMMsshallfollowtheseguidelinesand
shouldbeunderthesupervisionorguidanceoftheBiosafetyCommitteeorrelated
agencies. Importofpathogenicbacteriaormicroorganisms/agents
classifiedas
riskgroup2orhighershallcomplywiththePathogensandAnimalToxinsActB.E.2525
(C.E.1982),thePathogensandAnimalToxinsAct(No.2)B.E.2544(C.E.2001),and
currentlythisacthasenforcedforapprovalbytheDepartmentofMedicalSciences,
MinistryofPublicHealth(MOPH).ItmustalsocomplywiththeBiosafetyAct(under
review)(Appendix12).
2.
Importviainternationalpostshalladheretotheguidelinesdefinedby
theUniversalPostalUnion regardingnon-infectiousand
infectiousperishable
biologicalsubstances(NIH2002).
3.ThepossessionoruseofGMMsclass4isprohibited.
4.ForliveGMMtransport,thecontainersizeshallbe2timeslargerthan
theGMMvolume.InthecaseoftransportationofGMMclasses2and3involumes
greaterthan10litres,permissionmustfirstbegrantedbytheIBC.
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 33
Figure 10.1
Exampleofthetriplepackagingsystemforthepackagingandlabeling
ofCategoryA* infectious substances (modified from:Guidanceon
RegulationsfortheTransportofInfectiousSubstances,WorldHealth
Organization,2013)
*
CategoryAmaterialisaninfectioussubstancethatistransportedinaformthatiscapableofcausingpermanent
disabilityor life-threateningor fataldisease
tootherwisehealthyhumansoranimalsuponexposure.
Anexposureoccurswhenaninfectioussubstanceisreleasedoutsideof
itsprotectivepackaging,resultingin
physical contactwithhumansoranimals. (Source:Biosafety
inMicrobiologicalandBiomedical Laboratories
5thEdition,2009,p.340).AlistofinfectioussubstancesincludedinCategoryAisshowninAppendix13.
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34 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
Figure 10.2
Exampleofthetriplepackagingsystemforthepackagingandlabeling
ofCategoryB infectious substances (modified from:Guidanceon
RegulationsfortheTransportofInfectiousSubstances,WorldHealth
Organization,2013)
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 35
Figure 10.3 Exampleof the triplepackaging system forGMMs
(GILSP/class1)
(courtesyoftheBIOTECCultureCollection(BCC),NationalCenterfor
GeneticEngineeringandBiotechnology(BIOTEC),Thailand)
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36 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 37
Appendix 1Non-Genetically Modified Microorganisms
Microorganisms/agentsclassifiedasnon-GMMsandthereforeexcludedfrom
theseguidelinesarelistedbelow:
1.1Microorganisms/agentsmodifiedbymutagenesismethodsthatdonot
involveintroductionofnon-homologousDNA.
1.2Microorganisms/agentsgeneratedby
inductionofpolyploidismand
haploidism.
1.3Microorganisms/agentsgeneratedbyprokaryoticcellfusion(including
protoplastfusion)withoutintroductionofnewgeneticmaterial.
1.4Microorganisms/agentsgeneratedbyeukaryoticcell fusion
(including
protoplastfusion)withoutintroductionofnewgeneticmaterial.
1.5Microorganisms/agentsgeneratedby in vitrofertilization
1.6GILSPandClass1microorganisms/agentsgeneratedby
self-cloning.
Applicantsmust still submit theapplication form (Appendix8) to
confirm
theself-cloningwork.(Workwithclass2and3microorganisms/agentsshouldfollow
theseguidelines.)
[Self cloningmeans the removalofnucleicacid sequences
fromacellof
anorganismwhichmayormaynotbefollowedbyreinsertionofallorpartofthat
nucleicacid(orasyntheticequivalent),whetherunalteredoralteredbyenzymatic
ormechanicalprocesses,intocellsofthesamespeciesorphylogeneticallyclosely
relatedspecies(speciescapabableofhybridizingnaturally;seeTableA1.1).Thismay
include theuseof recombinantvectors,withanextendedhistoryof
safeuse in
aparticularorganism, tomanipulateand reinsert thenucleicacid
sequences,
butthevectorsshallnotconsistofanygeneticelementsotherthanthosedesigned
forvectorstructure,vectorreplication,vectormaintenanceormarkergenes.]
1.7Microorganisms/agentsgeneratedbyconjugation,
transformation,
transductionandsimilarnaturalprocessesasshowninTableA1.1
Remark: Classificationofmicroorganisms/agents intomicrobial
groups inAppendix1mustbeconsidered
onacase-by-casebasisbytheTBC.
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38 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
Table A1.1:Examplesofmicroorganisms/agents capableofnaturalDNA
transfer
withinthesamesublist.
Sublist Name
SublistA Genus Escherichia
Genus Shigella
Genus Salmonella - including Arizona
Genus Enterobacter
Genus Citrobacter - including Levinea
Genus Klebsiella - including K. oxytoca
Genus Erwinia
Pseudomonas aeruginosa, Pseudomonas putida,
Pseudomonas fluorescens and Pseudomonas mendocina
Serratia marcescens
Yersinia enterocolitica
SublistB Bacillus subtilis
Bacillus licheniformis
Bacillus pumilus
Bacillus globigii
Bacillus niger
Bacillus natto
Bacillus amyloliquefaciens
Bacillus aterrimus
SublistC Streptomyces aureofaciens
Streptomyces rimosus
Streptomyces coelicolor
SublistD Streptomyces griseus
Streptomyces cyaneus
Streptomyces venezuelae
SublistE One-waytransferof Streptococcus mutans
or Streptococcus lactis DNA into Streptococcus sanguis
SublistF Streptococcus sanguis
Streptococcus pneumoniae
Enterococcus (Streptococcus) faecalis
Streptococcus pyogenes
Streptococcus mutans
Remark: This listofmicroorganisms/agents capableofnaturalDNA
transferwithin the samesublist
mayberevisedonthebasisofscientificevidence.
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 39
Appendix 2Elaboration of criteria for GILSP
(Good Industrial Large Scale Practice) GMMs
TheclassificationofGMMs into theGILSPcategory isbasedonclear
demonstrationthattheGMMsusedaresafeandposenopossiblehazardtohumans.
Thecriteriausedtodeterminesafetyincludethenaturesofthehostcell,vectorand
insertedDNA/genes,andtheGMMsthemselves.
2.1 Host
Hostsused forpreparationofGILSPGMMsshallmeet the
requirements
statedin2.1.1–2.1.3or2.1.4.
2.1.1Non-pathogenic
The identityof thehostmustbeestablishedand the taxonomywell
understood.Thehostmustbeevaluated todetermine that it
isnotpathogenic.
ThehostshouldnotappearinlistsofhumanpathogensofWHOand/orNIH(USA).
Incaseswhereuncertaintyremainsforthepotentialpathogenicityofanorganism
oranattenuatedstrain,furtherdatamustbeprovidedtoassessitssafetyandhence
itssuitabilityforhandlingunderGILSPconditions.Inaddition,someorganismsthat
arenot found inpathogen listsmayproduce toxic substances
inamountswhich
requirefurtherevaluation.
ExamplesofhoststhatarecurrentlyusedinGILSPpracticearelistedbelow.
•Bacteria •Fungi
Bacillus subtilis Aspergillus niger
Corynebacterium flavum Aspergillus oryzae
Escherichia coli K-12
•Yeast •Cell lines
Candida boidinii Chinesehamsterovarycellline
Pichia pastoris Spodoptera frugiiperda cell line
Saccharomyces cerevisiae
Trigonopsis variabilis
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40 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
2.1.2Noadventitiousagents
Thisismainlyrelevanttocellcultureswhereharmfulmicroorganisms/agents,
inparticularharmfulvirusesandmycoplasma,shouldnotbepresentatdetectable
levels.Bacterialculturesshouldnotcontainunwantedphages.
2.1.3Extendedhistoryofsafeuse
There shouldbeadequateanddocumentedexperienceof safeuseof
thehostorganismandlackofharmtohumansandtheenvironment.Historicaland
other data on the host, its progenitors or closely related
strains may be appropriate
forevaluation.Suchevidencemaybeobtainedfromapplicationssuchasproduction
offood,enzymeandantimicrobialagents,includingdatafromdischargepractices
usedwithsuchapplications.Laboratoryuseand/orpilotscalefermentationunder
conditionsofminimalcontainmentcouldalsoprovideusefuldata.
2.1.4Built-inenvironmentallimitationspermittingoptimalgrowthinindustrial
settingbutlimitedsurvivalintheenvironment
Thepossibilityofadverseeffects canbe reducedby
restrictionson
theorganism’sabilitytomultiply,disseminateorsurvive.Thiscanbeachievedby
usingbuilt-instablebiologicallimitationswhich,withoutinterferingwithgrowthin
thebio-reactor,diminish
survivabilityandpreventadverseconsequences to
theenvironment.Examplesoforganismswithbiological limitations
include
auxotrophic strains,asporogenic strains,and strainswithbuilt-in
sensitivity to
environmentalfactorssuchasUVlight.
2.2 Vector and Inserted DNA or gene
VectorsandinsertDNA/genesintheGILSPcategoryshallmeettherequirements
below:
2.2.1Well-characterisedandfreefromknownharmfulsequences
- Vectors canbecharacterisedbya combinationof reference to
theliteratureorvariousotherlistings,aknowledgeofthederivation
andconstructionof thevector,and subsequentexperimental
confirmationoftheconstruct.Thecharacterisationshouldensure
thatthevectorisfreefromsequencesthatmayharmfultohumans
ortheenvironment,suchassequencesthatenableproductionof
substanceswhichcanhaveharmfuleffects,suchastoxinsorfactors
knowntobeinvolvedinpathogenicityand/orcolonisation.
- InsertedDNAorgenesmustbe identifiedas to their source,
positioning, function,andassociatedgenetic
sequencesaffecting
geneactivity,suchaspromoters,terminatorsandintrons.Inaddition,
insertDNAorgenesshouldnotharmfultohumansortheenvironment.
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 41
2.2.2Aslimitedinsizeaspossiblewhilemaintainingtheintendedfunction,
andshouldnotbeabletomaintainitselfintheenvironment.
2.2.3Shouldbepoorlymobilisable
Oneconsiderationarisingfromtheuseofvectorstointroduceaninsertis
therateatwhichthevector/insertcansubsequentlybetransferredfromtheoriginal
recipient.Forexample,therateofexchangeofplasmidvectorscanbeloweredby
theeliminationoftransferfunctions.Otherapproachescanalsobeusedtoreduce
thefrequencyatwhichtheinsertedDNAcanbetransferredfromtherecipientto
otherorganisms,throughmeanssuchasstableintegrationintothechromosome.
2.2.4Shouldnottransferanyresistancemarkerstomicroorganisms/agents
notknowntoacquirethemnaturally.
Frequently,genesforresistancetoavarietyofsubstances(e.g.antibiotics,
heavymetals)areintroducedintorecombinantorganismsforselectionpurposes.
Considerationsforevaluatingaspecificresistancegeneincludethefrequencythat
resistancemarker(s)canbetransferredfromtherecombinantorganismtoother
organisms,andwhethersuchacquisitioncancompromisetheuseofatherapeutic
agentor lead toenvironmentalperturbations.Markers for substances
suchas
antibiotics thatarenot currently in commercialuse
shouldalsobeevaluated to
determinewhether themarkerexhibits cross-reactivityor linked
resistance.
Furthermore,whetherselectionpressurefortheresistancemarkermightexistin
naturemustalsobeassessed.Forexample,environmentalselectionforanorganism
carryinga resistancegenemaybeenhanced if theselectingagent
inquestion is
presentinadequateconcentrationsintheenvironmentasaresultofantibioticuse
inlivestockfeedorpollutionbyenvironmentalcontaminantssuchasheavymetals.
2.3 GMMs
2.3.1Non-pathogenic
Thenatureand,whereappropriate,thesourceoftheinsertedgenesmustbe
considered.The typeofgeneproductand its functionmustbeexamined
in
thecontextofthecharacteristicsofthehost.If,forinstance,thegeneproducthas
noknownroleinpathogenicityandthehostisnotpathogenic,thentheGMMis
expectedtobenon-pathogenic.
2.3.2Assafeinindustrialsettingsasthehostorganismorwithlimitedsurvivalin,
andwithoutadverseconsequencesto,theenvironment.
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42 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
Ingeneral,theapproachtakenshouldbetoconsiderthenatureofthehost
andtofocusonthenatureoftheinsertedgenesandtheresultingproducts.Their
effectsonbiologicalfitnessandadaptability,includingattributessuchastheability
tocolonisenewniches,shouldbetakenintoaccount.Adverseconsequencescanbe
avoided,forexample,byusingGMMsoflimitedsurvivalintheenvironmentinrelation
tothewild-typestrain.Insomecases,itmaybenecessarytogenerateand/orcollect
dataonspecificproperties,forexample,throughmonitoringofenvironmentaldischarges.
GMMswiththesecharacteristicscanbeclassifiedintheGILSPcategory,andwork
withtheseGMMscanfollowGoodMicrobiologicalPracticeforPilotandIndustrialscales.
However,casesthatdonotfitthesecriteriauponriskassessmentbuthaveenough
datatosupportanassessmentoflowestriskcanbeconsideredonacase-by-casebasis.
Table A2.1:SuggestedcriteriaforGILSP
Topic Criteria for evaluation
Host - Non-pathogenic
- Noadventitiousagents
- Extendedhistoryofsafeuse
Or
- Built-inenvironmentallimitationspermittingoptimalgrowthin
industrialsettingbutlimitedsurvivalwithoutadverseconsequences
intheenvironment
Vector/Insert - Detailofhistory
- Well-characterisedandfreefromknownharmfulsequences
- LimitedinsizeasmuchaspossibletotheDNArequiredtoperform
theintendedfunction
-
Shouldnotincreasethestabilityoftheconstructintheenvironment
(unlessthatisarequirementoftheintendedfunction)
- Shouldbelowmability
- Shouldnottransferanyresistancemarkerstomicroorganisms
notknowntoacquirethemnaturally
GMMs - Non-pathogenic
- Assafeinindustrialsettingashostorganism,orwithlimited
survivalin,andwithoutadverseconsequencesto,theenvironment
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 43
Appendix 3List of safe host systems
3.1 Safe host/vector systems approved by the TBC
Category Host Vector
Bacteria 1. Agrobacterium radiobacter 1.
Non-tumorigenicdisarmedTiplasmid
Agrobacterium rhizogenes— vectors,orRiplasmidvectors
disarmedstrains 2. None(non-vectorsystems)
Agrobacterium tumefaciens —
disarmed strains
2.Bacillus subtilis Host-Vector 1 Systems*
Thefollowingplasmidsareaccepted
asthevectorcomponentsofcertified
B. subtilissystems:pUB110,pC194,
pS194,pSA2100,pE194,pT127,
pUB112,pC221,pC223,andpAB124.
B. subtilisstrainsRUB331andBGSC
1S53havebeencertifiedasthehost
componentofHost-Vector1systems
based on these plasmids
Host-Vector 2 Systems**
Theasporogenicmutantderivativeof
Bacillus subtilis,ASB298,withthe
followingplasmidsasthevector
component:pUB110,pC194,pS194,
pSA2100,pE194,pT127,pUB112,pC221,
pC223,andpAB124
3.Bacillus—specifiedspecies: 1. Non-conjugativeplasmids
asporogenicstrainswith 2. Plasmidsandphageswhosehost
areversionfrequencyof rangesdoesnotincludeB. cereus,
lessthan10-7 B. anthracis or other pathogenic
a)B. amyloliquefaciens strainsofBacillus
b)B. licheniformis 3. None(non-vectorsystems)
c)B. pumilus
d)B. subtilis
e)B. thuringiensis
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44 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
Category Host Vector
4.Escherichia coli (EK2) Plasmid Systems (E. coli
K-12strainchi-1776) Thefollowingplasmidsarecertifiedfor
use:pSC101,pMB9,pBR313,pBR322, pDH24,pBR325,pBR327,pGL101,and
pHB1.ThefollowingEscherichia coli/
S.cerevisiaehybridplasmidsarecertified
asEK2vectorswhenusedinEscherichia
colichi-1776orinthesterileyeaststrains
SHY1,SHY2,SHY3,andSHY4:YIpI,YEp2, YEp4,YIp5,YEp6,YRp7,YEp20,YEp21,
YEP24,YIp25,YIp26,YIp27,YIp28,YIp29, YIp30,YIp31,YIp32,andYIp33
Bacteriophage Systems ThefollowingarecertifiedEK2systems
basedonbacteriophagelambda: Vector Host lgt WESlB' DP50supF lgt
WESlB* DP50supF lgt ZJ virlB' Escherichia coliK-12 lgtALO.lB'
DP50supF Charon3A DP50orDP50supF Charon4A DP50orDP50supF Charon16A
DP50orDP50supF Charon21A DP50supF Charon23A DP50orDP50supF
Charon24A DP50orDP50supF
Escherichia coli K-12strainschi-2447
andchi-2281arecertifiedforusewith lambdavectors thatarecertified
for usewith strainDP50orDP50supF providedthatthesu-strainnotbeused
asapropagationhost 5.Escherichia coliK-12,E.coliBor
1.Non-conjugativeplasmid E. coliCorE. coliNissle1917-
2.Bacteriophagethatarelambda, anyderivativethatdoesnotcontain
lambdoid,andFdorF1,suchasM13 a)generalizedtransducing
3.None(non-vectorsystems) phages; or b) genesabletocomplement
theconjugationdefectina non-conjugativeplasmid
-
Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales 45
Category Host Vector
6. Lactobacillus 1.Non-conjugativeplasmids Lactococcus lactis
2.None(non-vectorsystems) 7. Oenococcus oenisyn.
1.Non-conjugativeplasmids Leuconostoc oeni
2.None(non-vectorsystems) 8. Pediococcus 1.Non-conjugativeplasmids
2.None(non-vectorsystems) 9. Photobacterium angustum
1.Non-conjugativeplasmids 2.None(non-vectorsystems)
10.Pseudoalteromonas tunicata 1.Non-conjugativeplasmids
2.None(non-vectorsystems) 11.Pseudomonas putida-strain
1.Non-conjugativeplasmids, KT2440 includingcertifiedplasmids
pKT262,pKT263,pKT264 2.None(non-vectorsystems)
12.Rhizobium(including 1.Non-conjugativeplasmids Allorhizobium)
2.None(non-vectorsystems) 13.Sphingopyxis alaskensissyn.
1.Non-conjugativeplasmids Sphingomonas alaskensis
2.None(non-vectorsystems) 14.Streptococcus thermophilus
1.Non-conjugativeplasmids Synechococcus—specifiedstrains:
2.None(non-vectorsystems) a)PCC7002 b)PCC7942 c)WH8102
15.Streptomyces—specifiedspecies: 1.Non-conjugativeplasmids a)S.
aureofaciens 2.Certifiedplasmids:SCP2,SLP1, b)S. coelicolor
SLP2,PIJ101andderivatives c)S. cyaneus
3.ActinophagephiC31andderivatives d)S. griseus
4.None(non-vectorsystems) e)S. lividans f)S. parvulus g)S. rimosus
h)S. venezuelae 16.Synechocystisspecies-strain
1.Non-conjugativeplasmids PCC680316. 2.None(non-vectorsystems)
17.Vibrio choleraeCVD103-HgR 1.Non-conjugativeplasmids
2.None(non-vectorsystems)
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46 Biosafety Guidelines for Contained Use of Genetically
Modified Microorganisms at Pilot and Industrial Scales
Category Host Vector
Fungi 1. Kluyveromyces lactis 1.allvectors
2.none(non-vectorsystems) 2. Neurospora crassa Host-Vector 1
Systems* Thefollowingspecifiedstrainsof Neurospora
crassawhichhavebeen modifiedtopreventaerialdispersion:
In1(inositol-less)strains37102,37401,
46316,64001,and89601.Csp-1strain UCLA37andcsp-2strainsFS590,
UCLA101(theseareconidialseparation mutants).
EasstrainUCLA191(an"easilywettable" mutant). 3. Pichia pastoris
1.allvectors 2.none(non-vectorsystems) 4. Saccharomyces cerevisiae
Host-Vector 2 System** Thefollowingsterilestra