at pilot and industrial scales - biotec...containment at pilot and industrial scales according to...

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


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

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

ii 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 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

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

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

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

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.

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.

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.

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.

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

xii 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 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

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]


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


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).


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.


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.


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.


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.


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.


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.


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.


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.


8. Onlypertinentoperatorsmayhaveaccesstocontainedareas.

9.Safetyplansandemergency trainingmustbeprovided forpertinent

operators so that theymanageemergency situations suchasGMMspillageor

contamination.Emergencyprotocolsmustbepostedinworkingareas.

10.Emergencyequipmentandtoolsmustbelocatedinworkingareasand

routinelycheckedtoensurethattheyareingoodconditionatalltimes.

11.Signsdisplayingcontainmentlevelsmustbepostedinthecontainedareas

andonequipmentforGMMuse.IncidenceofspillageorreleaseofGMMsmustbe

reportedtotheIBCandTBCimmediately.


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.


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.


Chapter 5Approval Process for Projects with Contained Use of GMMs


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.


Figure 5.1 TheapprovalprocessflowchartforprojectswithcontaineduseofGMMs

atpilotandindustrialscales


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.


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.


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.


- 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.


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.


3. InthecaseofexposuretoGMMsclassifiedasclass2or3,intensivemedical

check-upsbyqualifiedphysiciansaswellasbloodtestsandfollow-upsonsymptoms

oreffectsofdiseasesmustbeconducted.

4. Inthecaseofworkwithclass3GMMs,operatorbloodsamplesmustbe

drawnpriortocommencingGMMworkandkeptforatleast10yearsaftercompletion

oftheworktoallowmonitoringforcausesofsicknessordiseasethatmaysubsequently

develop.


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.


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)


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.


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


Table 8.2:Wastemanagementrequirements

•

•

•

•


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.


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.


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.


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.


Figure 10.2 Exampleofthetriplepackagingsystemforthepackagingandlabeling

ofCategoryB infectious substances (modified from:Guidanceon

RegulationsfortheTransportofInfectiousSubstances,WorldHealth

Organization,2013)


Figure 10.3 Exampleof the triplepackaging system forGMMs (GILSP/class1)

(courtesyoftheBIOTECCultureCollection(BCC),NationalCenterfor

GeneticEngineeringandBiotechnology(BIOTEC),Thailand)


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.


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.


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


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.


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.


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


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



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



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)



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