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Biosafety in the laboratory

1. 8 Transfer of material to an autoclave situated outside the laboratory should occur accordin equivalent protection level to a validated method that guarantees an Containment requirements he bil hard 52 3 Working practices and waste management for the containment zone Specifications Containment level Al A2 A3 A4 34 Restricted access required required required and control required and control 35 Notification on the door Biohazard required except the required required required symbol coordinates of person in Biohazard symbol charge containment level type of biological risk list of persons who have access criteria for admittance 36 Animal housing to contain its own not required recommended required required equipment 37 Protective clothing that is specific required required required optionally required complete appropriate shoe change of clothing for experimentation 54 In case of manipulation of zoopathogens period during which contact of the personnel with the host animals should be avoided not required protection and footwear when entering or leaving 38 Decontamination of the protective not required not required required required clothing before it leaves the containment zone 39 Gloves optional recommended required required 40 Mouth mask not required optional optiona
2. Eimeria acervulina Eimeria burnetti Eimeria maxima Eimeria necratix Eimeria spp Leishmania brasiliensis Leishmania donovani Leishmania ethiopica Leishmania major Leishmania mexicana Leishmania peruviana Leishmania spp Leishmania tropica Plasmodium falciparum Plasmodium spp with humans and apes Pneumocystis carinii Theileria annulata Cryptococcus neoformans Fusarium coccophilum Penicillium marneffei Pneumocystis carinii Rhizomucor pusillus Rhizopus cohnii Rhizopus microspous Theileria hirei Theileria mutans Theileria ovis Theileria parva Theileria taurotragi Toxoplasma gondii Trichomonas foetus Trichomonas vaginalis Trypanosoma brucei brucei Trypanosoma brucei gambiense Trypanosoma brucei rhodesiense Trypanosoma congolense Trypanosoma cruzi Trypanosoma equiperdum Trypanosoma evansi Trypanosoma vivax Note This is a shortened list of pathogens If your organism is not on this list first consult the lists at www biosafety be before concluding that your organism is not pathogenic Fytopathogens On the following page the risk group of some relevant fytopathogens is given Some fytopathogens however are also subject to additional quarantine regulations that are designed to combat relevant plant diseases or diseases that can affect plant products Biosafety in the laboratory Fytopathogenic viruses P Alfalfa mosaic virus Apple chlorotic leaf spot virus Apple mosaic virus Apple s
3. If you have general questions about classifications or necessary containment measures you should first contact your superior And in case this does not give you a decisive answer you should subse quently contact your groupleader internal biosafety expert or external biosafety expert he Responsible persons and sources 69 Lose des 70 Question 1 Question 2 Question 3 Question 4 Question 5 Question 6 Question 7 Question 8 Question 9 Question 10 Question 11 Question 12 Question 13 Question 14 What is the definition of a genetically modified organism How many biological risk classes or risk groups are there to classify the ability of orga nisms to cause disease Mention three types of hazards that are potentially associated with living and or modi fied organisms To what risk group or biological risk class belongs wild type E coli To what risk group or biological risk class belong E coli K12 strains Describe the different steps of the risk assessment process for genetically modified orga nisms What is an aerosol In what way can the combination of an immortalised cell line with a viral vector give rise to the unwanted spread of genetically modified material Mention four important physical containment measures True or false the culture fluid containing genetically modified micro organisms of risk class 1 can be thrown down the sink after the exper
4. The plants that are not able to survive in the ecosystem for instance banana The plants that have no relatives in the ecosystem with which they could hybridise successfully for instance banana The plants that have been infected by a virus of biological risk class 2 by a genetically modified virus of risk class 1 or 2 by a viral vector of biological risk class 1 or 2 or that carry a viral genome of biolo gical risk class 1 or 2 The plants that carry a non selftransmissible fytopathogen of biological risk class 1 or 2 Containment level G2 The crosspollinating selfpollinating windpollinating and insectpollinating plants The plants whose full life cycle can only take place in the ecosystem and whose seeds can survive for long periods of time depending on the case and the experience The plants that have been infected by a virus of biological risk class 3 by a viral vector of biological risk class 3 or that carry a viral genome of biological risk class 3 The plants that carry a selftransmissible fytopathogen or a GMO of biological risk class 2 or a fytopa thogen or a GMO of biological risk class 3 Containment level G3 The plants that have similar properties as described in level G2 but that carry a hazardous transgene or a virus of biological risk class 4 or a viral genome of biological risk class 4 The plants that carry genes that are the subject of first tests of gene flow and that originate f
5. What constitutes a hazardous gene product is the most difficult risk classification question Virulence gene products might be an example of a hazardous gene product Final risk classification depends on the suspected effect of the gene product in the used hostorganism in 58 Biosafety in the laboratory 2 Activities with animal cells without use of viral vectors and with the use of baculoviruses c 2 Sons sE a EE 2RTRSIII oo EER 5 First direction I S F j ESSE EES 3 Eg ease 5 Se EeHESS ZEEZELEE 9 E SBREBEBRSI SBBBELIS v T 12 13 il 12 Li 12 3 l4 l4 13 13 E 12 12 E E 2 12 Ibs 5 12 12 Li 12 Z E 5 z E z 12 i 13 EZ Bie 12 12 s LI 12 28 Li 12 f The sequence contains genetic 12 13 information that codes for the production li 12 of a toxin of respectively risk group Li 12 g The sequence contains genetic 14 14 information for the formation of a Risk group 3 JEN 13 for eucaryotic cells infectious virus Risk group 2 iy D E of respectively risk group 4 3 or 2 and the viral sequences that have 2 been brought into the host can give rise to the formation of autonomously 5 E replicating virusparticles h The sequence contains genetic 12 13 gt s information for the formation of a defect Risk group 3 iW 12
6. b A T 2 toxin is a toxin with an LD50 for vertebrates between 100 nanograms and 1 microgram per kg bodyweight c A T 1 toxin is a toxin with an LD50 for vertebrates between 1 microgram to 100 micrograms per kg bodyweight Requirements for host vectorsystems to be classified in risk class 1 Requirements for smallscale laboratory activities lt 100 liter effective culture volume 1 The host is not pathogenic 2 The host is free of known biological agents that are potentially hazardous 3 The vector is well characterised this is the type of vector should be defined plasmid cosmid mini chromosome etc the size should be known function and origin of structural genes and markerge nes should be known as well as restrictionsites and replicon Commercially available vectors are mostly well characterised 4 The vector may not have hazardous consequences may not transfer virulence or toxindetermining elements The vector is limited to only necessary elements as far as possible The vector may not advance the stability of the GMO in the environment The vector should be difficult to mobilise Tra The vector is not allowed to pass on resistance genes to micro organisms that do not posses these genes naturally if the uptake of such a resistance gene by this organism would disable the use of antibiotics in healthcare OND U Ra TES With organisms of risk groups 4 3 and 2 is meant the organisms that ha
7. Feline sarcoma virus FeSV Human immunodeficiency viruses HIV types 1 amp 2 Human T cell lymphotropic viruses HTLV types 1 amp 2 Leukomogenic murine oncovirus Murine lymphosarcoma virus MuLV Lymphosarcoma viruses of nonhuman primates Monkey mammary tumor viruses MPTV Murine mammary tumor viruses MMTV Murine sarcoma viruses MuSV Porcine sarcoma virus Rat lymphosarcoma virus Rat LSA Reticuloendotheliosis viruses REV Simian foamy virus Simian immunodeficiency viruses SIV Simian sarcoma viruses SSV Note This is a shortened list of pathogens If your organism is not on this list first consult the lists at www biosafety be before concluding that your organism is not pathogenic The risk groups of some relevant pathogens 65 66 Rhabdoviridae Rabies virus Vesicular stomatitis virus VSV Togaviridae Semliki Forest virus Sindbis virus Rubella virus Not classified Blood borne hepatitis viruses not identified yet Borna diseases virus Human and animal pathogenic fungi H A Aspergillus flavus Aspergillus fumigatus Aspergillus nidulans Aspergillus parasiticus Aspergillus terreus Aspergillus versicolor Candida albicans Human and animal pathogenic parasites H A Unrelated agents connected with Bovine spongiform encephalopathy BSE Chronic wasting disease of deer Creutzfeldt Jakob disease Gerstmann Str ussler Scheinker syndrome Kuru Mink encephalopathy Scrapie
8. with the outside world manual operation VENTILATION 17 Input airflowsystem separated from not required not required recommended required adjacent laboratories Extract airflowsystem separated from required not required recommended required adjacent laboratories Input and extract airflowsystem required not required required required interconnected to prevent accidental overpressure Input and extract airflowsystem can be required not required required required closed using valves Negative airpressure in the controlled required not required required control and required control zone when compared to adjacant zones alarm systems and alarm systems 22 HEPA filtration of the air not required not required required on the required on the extract air input airflow and double filtration on the extracted air 23 System that allows changing of the required required filters without contamination of the environment 24 HEPA filtered air may be recirculated optional no 25 Specific measures to ventilate to such optional optional required required an extent that contamination of the air is minimised 1 In case of use of virus not retained by HEPA iltration special appropriate measures must be taken with regard to the extract air Measures to be specified in the notification and the competent authorities should determine the requirements in the authorisation 2 Safety equipment Specifications Contai
9. In practice to prevent unauthorised personnel from gaining access the laboratory should be kept locked when nobody is working inside For level 3 and 4 activities this is an absolute requirement Authorisation to work in level 2 3 and 4 laboratories should only be given when the labo ratory worker has proven that he has sufficient knowledge of safe working practices that he has learnt all the specific safety procedures for this particular laboratory and knows how to apply them There should be records of authorised personnel which should be updated on a regular basis To prevent unauthorised access level 2 and especially level 3 and level 4 laboratories should not be located near the entrance or exit of floors or buildings or in areas where many people circulate Biosafety in the laboratory 6 CONTAMINATION ACCIDENTS DECONTAMINATION AND A FON Minor contamination incidents can happen sooner than one might think Some fluid may be spilled when it is being poured out or some droplets may splash It is important to deal with contaminations immediately Other accidents like the breaking of glassware needle prick or cutting accidents may also happen before you know it These different kinds of accidents always result in exposure to the organism There is no immediate risk when a harmless class 1 organism is involved but even in such cases it is necessary to clean and decontaminate Not surprisingly because if this is not done
10. constructions deri ved from viruses The use of viruses or viral vectors always implies the use of host cells Without host cells no virus can be replicated In practice there are three types of activity 1 the growing of cells to produce viral particles 2 the handling of viral particle contai ning supernatants for quality controls etc and 3 the transduction of a cell line test animal or plant Especially supernatants may contain very high levels of viral particles These supernatants should be handled carefully Once the cell animal or plant has been infected the danger depends on the virus or viral particle s ability to replicate In some The spread of organisms in the laboratory cases a replication defective virus is used which means that the virus can infect the cells but is no longer able to replicate The ability to spread or replicate may differ from one virus to another Some viral particles are able to spread through the air or to survive for very long periods of time Other viruses such as HIV are extremely vulnerable outside their host Plant viruses sometimes need vectors to be able to spread These vectors are often insects that suck up the virus and spread it to other plants Transgenic plants Transgenic plants are grown in vitro in growth chambers or greenhouses and the plants are not able to disseminate just like that Nevertheless the undesired spread of transgenic plants deserves special attenti
11. to adjacent zones 24 HEPA filtration of the air not required optional required on the required on the inward extracted air airflow and double filtration on the extraced air 25 System that allows changing of required required the filters without contamination of the environment 26 HEPA filtered air may be recirculated optional forbidden 21 Specific measures to ventilate to such optional optional required required an extent that contamination of the air is minimized In case of use of virus not retained by HEPA filtration special appropriate measures must be taken with regard to the extract air Measures to be specified by the notifier and by the competent authorities 2 Safety equipment Specifications Containment level Al A2 A3 A4 28 Microbiological safety cabinet not required optional optional optional class 1 or Il class 1 or 11 class IIl or class ll with the use of an isolation suit fitted with overpressure 29 Animals in cages or equivalent optional optional optional optional isolation installations 30 Cages fitted with a HEPA filter not required optional required required 31 Autoclave on site in the building in the animalarium or in the animalarium an adjacent room 32 Double ended autoclave not required not required recommended required 33 Fumigationsystem or not required recommended required required decontamination immersion bath
12. 31 lID S17 I I dno4b 3su 01 Buo oq u asvasip asnvo o 3 qp Aabuo ou PUV pajunuajjD 340 SUWAS ZIY 1l03 7 asvasip ISNDI 0 3 QD a40f24211 S1 IJ Z dno4D ysu si 110273 Ad PUM S D2uD DQ 1031601033 fo 32uvqunjsip 40 amp 121x0 Apioiuaboyjod aouvjsui 404 3spasip ISNDI UDI DY SUSIUD AO 340 p pun Z dnoar aspasip asnvo 0 3 qD jou aap suisiuvb1o dnoar p 01 sdno4 ysu anof aan 34911 UOYDUIQUIOIIA qp4njpu 40 uoijonpoadaa fo Suwaw Aq ajqissod jou st joys om D UL po43 U uaaq SVY Pupu JYIUIH ai Yot ui wstunbro uv si wsiubbuo parfipour amp mouauab v pI uonsan SET uonsan zI uotnsan ITT uonysan 01 uoysand 6 uojsan g uoysand Z uoysand 9 uoysand G uoysand F uonsan uonsan Z uonsan uoysand Selftest Lose de 71
13. as much as possible When working with organisms that hold a certain risk starting from risk class 2 one should perform aerosol producing activities in a safety cabinet Pouring fluids Falling droplets Emptying a pipette by blowing Opening of wet caps Centrifugation by means of open tubes Inoculation needles that are too hot KERIS Figure 3 aerosolproducing procedures Undesired spread of organisms or genetic material It may have become clear that the spread of hazardous organisms represents a danger both to yourself and to your colleagues When it is possible for organisms to spread to a colleague they may spread to the environment as well This dissemination of organisms or genetic material to the environment is often undesired since it may involve the spread of The spread of organisms in the laboratory f nt ag 16 pathogens or toxins or lead to the disruption of ecological balances This is undoubtedly true with regard to organisms belonging to risk classes 2 3 and 4 However even the spread of organisms and their genetic material belonging to risk class 1 and thus presen ting only a minor risk should be limited Bacteria yeasts and fungi Bacteria are often capable of transferring genetic material This is especially the case when vectors are used that are self transmissible In practice to avoid genetic material from being easily transferred vectors are usually used that are difficult to mob
14. be inactiva ted or decontaminated Living coli bacteria must never end up in the residual waste Clearly marked waste bins for biological waste should therefore be present and fluids should be sterilised before they are poured out in the sink Alternatively chlorine could be used to inactivate the materials but this is a less environmentally friendly option An exception to this rule is E coli K12 strains carrying a non mobilisable mob and well characterised vector a fully characterised insert with regard to its origin size gene pro duct function s etc and producing a fully known non hazardous gene product Strains meeting these criteria do not need to be inactivated However these are very rare excep tions which hardly ever occur in research settings Moreover an explicit and specific authorisation stating that inactivation is unnecessary is always required Note It should be checked on a regular basis twice a year whether the used strains are still the pro per ones Alternatively the strains can be replaced regularly by new ones from a clean stock Risk assessment example gt E W 36 As an illustration of how a risk class and the necessary containment measures are determi ned and also how this booklet can be used some practical examples of risk assessment are given below Practical example of how to determine classification 1 Cloning a chymosin gene into a pUC18 using E coli JM109 as a host and p
15. case for E coli Sterilisation is the recommended method to this end Note Recently transformed little plants are often transferred onto fresh culture mediums This activity is often performed in a cross flow cabinet which is permitted as long as no open sources of Agrobacterium are involved Working with commonly used laboratory organisms Working with transgenic plants iu 44 Researchers working with transgenic plants should abide by the following rules of thumb 1 When working in fytotrons or growth chambers growth chambers should be closed and should not lead out into the open air plant material should be sterilised before it is discarded as residual waste in case the material still contains genetically modified A tumefaciens e the spread of seeds should be avoided and reproductive parts of plants should be inactivated before they are discarded as residual waste 2 When working in greenhouses when flowering insect pollinators are used all openings should be covered with insect screens and the greenhouse itself should not lead out into the open air an airlock should be present When non flowering plants or flowering self pollinators apo micts or wind pollinators are used insect screens are not required and an airlock is not really necessary In this case too the spread of seeds should be avoided Seeds should be carefully col lected and soil that might contain seeds should be inactivated before r
16. for eucaryotic cells infectious virus of Risk group 2 Li 12 E respectively risk group 4 3 or 2 and 5 the viral sequences that have been brought 5 into the host cannot give rise to the formation of autonomously replicating virusparticles 2 i The sequence contains genetic Li 12 3 information that codes for a hazardous gene product other than in j The sequence does not contain genetic Li 12 information that codes for a hazardous gene product What constitutes a hazardous gene product is the most difficult risk classification question Virulence gene products might be an example of a hazardous gene product Final risk classification depends on the suspected effect of the gene product in the used hostorganism ae Bre Guidelines for the classification of GMO activities 59 3 Activities with animal cells in combination with viruses or viral vectors 5 su g sgue tod 5 280585 ERSTE S Bees 4 s 585 ovx SOES 5 2 ISELESSBI Ishsiztreptf E 2g 2g PSS E PP vas esa Pe First direction 8m SSS SELEERBES gs gelsesgls geese sls S Pee 00558325 ss ESHES A nd ges Bago skeey STETE ES oi Tal MN Ee ESSEBSEE n a Resp L4 L4 L4 Resp L3 L3 L3 Resp L4 L4 L3 Resp L3 L2 L2 Resp 14 L4 L3 Resp L3 L2 L2
17. for the production of chymosin a cheese clotting enzyme is used It is well characterised known to be no toxin to have nothing to do with viruses or pathogenic organisms and to pose no threat in any other way either It can the refore be concluded that category j of the second direction of annex 2 chapter A is applicable here and that the corresponding containment level is L1 Step 4 Are there any special details with regard to 1 the environment that might be exposed to the materials 2 the type and level of the activities and 3 any non standard procedures In this particular case only small scale standard activities will be performed such as transformation small scale culturing of bacteria in Erlenmeyer flasks protein isolation gel electrophoresis An acti vity is only considered to be large scale when large volumes are being produced in production faci lities However even small fermenters require special attention as far as their leak tightness and the physical containment of the downstream processing are concerned In this case no special proce dures representing an additional risk or requiring a specific form of additional containment are performed The eventual containment level will therefore be standard L1 containment This booklet provides the following information on this topic L1 containment requirements annex 1 to this booklet Detailed SMP procedures chapter 5 of this booklet Decontamination and inactivati
18. 1 by destroying the lipid membrane of a micro organism which results in the leaking of cell material 2 by destroying proteins and enzymes necessary for the survival of the micro organism Ethanol quaternary ammonium salts and surfactants a o detergents and soap exploit the first mode of action Strong oxidising agents like chlorine of hydrogen peroxide exploit the second mode of action Phenolics like lysol work by destroying both proteins and the lipid membrane Some more information on specific disinfectants e Ethanol alcohol Ethanol is highly effective against vegetative bacteria fungi and viruses surrounded by a lipid membrane but not against spores Its effectiveness against non lipid viruses strongly varies Ethanol should be used as a 70 solution Mixtures of ethanol and other substances such as ethanol mixed with 100 g formaldehyde per liter or 2 g chlo rine per liter are more effective than ethanol alone Note that the degenerated alcohol used in laboratories is poisonous Solutions of ethanol deteriorate over time which is why they should be replaced at least once a month Biosafety in the laboratory Sodium hypochlorite bleach The chlorine released is a strong oxidising agent which is effective against all types of micro organisms Sodium hypochlorite must be used as a 20 ml l to 100 ml l solution depending on how dirty the circumstances are for instance a 1 in 10 solution of domestic bleach Solutions of
19. 5 Resp L4 L4 L4 Resp L4 L4 L4 Resp L4 L3 L3 Resp L3 L3 13 Resp L4 L3 L2 Resp L3 L2 L2 2 3 5 ES Resp 14 L3 L3 Resp 13 13 13 E Resp 14 13 L2 Resp 13 L2 L2 3 Resp L4 13 L2 Resp 13 L2 L1 E amp E s 5 Resp L4 L3 L3 Resp L3 L3 L3 26 Resp L4 L3 L2 Resp 13 L2 L2 E g Resp L4 L3 L2 Resp L3 L2 L1 e Resp 14 13 L2 Resp 13 L2 L1 lt s The sequence contains genetic Resp L4 L4 L4 Resp L3 L3 L3 information that codes for the production Resp L4 L4 L3 Resp L3 L2 L2 of a toxin of respectively class TI Resp 14 L4 L3 Resp 13 L2 L2 g The sequence contains genetic Risk group 4 Resp 14 14 14 Resp 14 L4 L4 information for the formation of a for Risk group 3 Resp 14 13 13 Resp 13 13 13 eucaryotic cells infectious virus of Risk group 2 Resp L4 L3 L2 Resp L3 L2 L2 respectively risk group 4 3 or 2 and the E viral sequences that have been brought into the host can give rise to the s formation of autonomously g replicating virusparticles E h The sequence contains genetic Risk group 4 Resp L4 L3 L3 Resp 13 13 L3 5 information for the formation of a defect Risk group 3 Resp L4 L3 L2 Resp L3 L2 L2 m J for eucaryotic cells infectious virus of Risk group 2 Resp L4 L3 L2 Resp L3 L2 L1 EE respectively risk group 4 3 or 2 and the viral sequences that have been brought 8 5 n r into the host cannot give rise to the i f
20. DGEMEN TER 68 ANNEX 5 RESPONSIBLE PERSONS AND SOURCES eed 69 ANNEX 6 SEURIES ICE E RTT RR EE 70 Contents Biological material is used intensively in biological and biomedical research In this type of research the use of modern molecular biological techniques like recombinant DNA tech nology is still on the increase This booklet gives a brief overview of the basic principles that are important for the safe use of pathogenic and or genetically modified organisms Three types of safety measures can be distinguished a measures to protect the worker b measures to protect the experiment and c measures to protect humans and the environment In this booklet these different measures will be discussed for micro organisms as well as for plants and animals Every researcher that makes use of these types of organisms in the laboratory should know the basic principles of biosafety by heart and act accordingly Only in this way a situation can be created that is safe both for yourself your fellow human beings and the environment The safe use of biological material is also required by two types of legislation 1 The legislation on the protection of workers of activities with biological agents 2 The legislation on the environment with regard to working with genetically modified organisms For both types there is European legislation which has been incorporated into national law Figure 1 The biohazard symbol This symbol
21. FLANDERS INTERUNIVERSITY INSTITUTE FOR BIOTECHNOLOGY T l wv I FLANDERS INTERUNIVERSITY INSTITUTE FOR BIOTECHNOLOGY 3rd revised edition May 2004 VIB publication Flanders Interuniversity Institute for Biotechnology Editor Ren Custers regulatory affairs manager VIB This booklet can be ordered from VIB Rijvisschestraat 120 9052 Zwijnaarde Belgium tel 09 244 66 11 fax 09 244 66 10 e mail vib vib be web http www vib be Responsible publisher Jo Bury VIB Rijvisschestraat 120 9052 Zwijnaarde Belgium Nothing from this publication may be copied and or made public for commercial purposes For educational purposes the source has to be mentioned May 2004 This is the third revised edition of the booklet Biosafety in the laboratory In this booklet you will find all the information that was already present in the earlier editions Only a few alterations have been made especially in the text concerning decontamination and inacti vation Modern biotechnology is a very important technology in the current biological and bio medical research Thousands of researchers in the life sciences are using micro organisms plants or animals on a daily basis to answer relevant scientific questions More and more use is made of genetically modified organisms The scientific community itself has been the first to stress the importance of working safe ly with GMOs Nowadays many regulations a
22. Y sjpuajmut pojputujuoo J DUu310d 3s D T gupunupjuooap AaYJOUD asn Pinoys nok 1990301307 asn 32unjsui sof nod fi og sasods jouajong 1suivDp affo jou st JOUDYII SAIMOYIND juajaduimo 21 fo uouvsuoypnv proydxa uv paau nok yoy sof ma su 0 suoidaoxo maf Quo 34D 34311 Psv sp papivosip aap Qu 34of59q pajvauovui aq pjnous swusiupb10 o1IWM WD I SSD ASL OS U 3s D J ya moyfiw papauno pauarf VAAH Yow uv aanssaud amw sayobau J uiqp5 Ajafos IT SSDII D Q1SD pajvunuvjuoo aq UDI jui sauouaq YJOOUS smopuim pup s10op pasojd aap sajduoxy uonpuiquio224 YHnoAY sajouavd snua snoyoafur fo uoyouuof y 01 asia amb Kow 109Daa y ut Juasasd saouanbos pua wa 4219501 1011 AJH JO Sub 393 IY UIDJUOD WITH 22unjsui sof saouanbas pa uijuoo vw 1129 pasijpiounut uy JUaMUOLAUA Iy 0 ppauds 0 swstupbuo asa sof suvom p apiao4d a40f042 pun suisiupBuo o421 2 qvia u1D u02 UDI sjojdo4p ASL AID ay via ppoads o 3 QD a4v y pinjf fo sjajdoap jjvwuis aa fo dn apou si joso420 uy SSD su puf y AuW F suoivaado pavpuvjs uou Kuv ajqvoiddo fi puo na 430 ai fo ajvos puo add aui usiupfuo ay 01 pasodxa aq pjnoo joy juaunuoa1au2 y fo s ui doid au junoooD Our AD Kqafos wsiunbio ay ajpuvi 0 AQV Iq 0 saansvaw JUIMUIIJUOI apssao2u ay amp fiuapi SSO su su woul Buiuvis c ssvjo ysu ajpudoaddo ay fo vopi suf p amp fiuapi 0 nok sajqpua sI Z 4034 puo y su soy fo sau42doad snopavzvi joyuajod
23. a pathogen it is very important to mention its host since infec tious diseases are an interaction between a pathogen and a host Some pathogens have a broad host range whereas others may only be able to infect one or a few hosts Moreover the risk group of a particular pathogen that can infect both humans and animals may differ from one host to another For instance the biological risk class of Herpes virus B is 3 for humans while it is 2 for animals There are published lists of the biological risk clas ses of different pathogens which are a convenient help for risk assessment In Belgium the following lists are important The pathogen classification list in Vlarem Il chapter 5 51 http www biosafety be and annex 1 to the Royal Decree of August 4 1996 concerning the protection of employees against the risks of exposure to biological agents at work this annex applies to human pathogens only Official Journal of Laws and Decrees in Belgium October 1 1996 Annex IIl of this booklet lists the biological risk classes of a number of relevant pathogens Opportunistic pathogens are organisms that are only able to cause disease in animals or humans which have an impaired immune system These organisms belong to biological risk class 1 There are no legal requirements for working with non genetically modified risk class 1 organisms It is however strongly recommended to apply the principles of Safe Microbiological Practices SMP when
24. al waste and or biological residues contaminated cadavers faeces using an appropriate and validated method before dumping required required required required 56 Inactivation of contaminated material glassware cages etc using an appropriate and validated method before reuse or destroying required required required required 57 Inactivation of the effluents of sinks and showers using an appropriate and validated method before dumping not required not required recommended required Faeces from transgenic animals does not have to be inactivated Biosafety in the laboratory 1 Equipment and technical requirements Belgian requirements for G1 G3 growth chambers and greenhouses Requirements for G4 greenhouses are not given in the Belgian legislation Specifications Containment levels G1 G2 G3 1 The greenhouse is a permanent structure not required required required 2 A 1 5m free zone in concrete or free of plant not required required required growth surrounding the growth chamber or greenhouse 3 A secured fence not require not required required 4 Aisles stabilised solid material solid material soil 5 Entry via a separated room with two interlocking not require optional required doors 6 Doors that can be locked not require required required 7 Structure resistant to shocks not req
25. and a higher level of containment will be required rule Belonging to Non selftransmissible L1 l eorj Most commercially E coli K12 vectors tra available systems BorC belong to this category Belonging to Selftransmissible 12 2 e or j An example is some E coli K12 vectors tra mini Tn5 systems Bor C Other E coli Non selftransmissible L2 3 e or j Unless proven strains vectors tra otherwise one should consider these hoststrains to be pathogenic on the assumption that non hazardous inserts are used hazardous inserts would require a higher risk class as applied in the schemes of annex 2 Working with commonly used laboratory organisms f ze 35 Laboratory requirements In many cases E coli K12 B or C strains are used in combination with non self transmissi ble vectors In such cases a basic L1 laboratory is sufficient The work surfaces should of course be easy to decontaminate Open handling of E coli on a bench is permitted provi ded that the creation of aerosols is minimised see SMP guidelines Inoculations can be performed near a flame Instead of working with open sources on a bench researchers can alternatively perform these operations in a safety cabinet The requirements of Safe Microbiological Practices should be met Waste All solide and pasty materials culture mediums all liquids containing modified coli bac teria and all materials that are possibly contaminated with coli bacteria should
26. at it will hybridise with wild relatives reproduc tive plant parts should be destroyed before they are discarded as waste pollen seeds stembase Figure 5 the reproductive parts of Brassica Biosafety in the laboratory The spread of transgenic plants or transgenic plant materials Potten Seeds Reproductive materials Arabidopsis In containment Seeds are very small upon No other than pollen Arabidopsis is a self pollinator touch they can be catapulted and seeds The spread of pollen is very into the air and spread very unlikely to have an impact easily Special aracons or other comparable means of containment should be used Tobacco Self pollinator The spread of No special remarks No other than pollen and pollen is very unlikely to have seeds an impact Rice Self pollinator The spread of No special remarks No other than pollen and pollen is very unlikely to have seeds an impact Tomato Insect self pollinator No special remarks No other than pollen and Pollinating insects should seeds be kept out Potato Self pollinator The spread of No special remarks The spread of tubers should pollen is very unlikely to have be prevented an impact Brassica Insect self pollinator No special remarks The spread of roots and Pollinating insects should stembases should also be be kept out prevented Maize Wind pollinator Spread of No special remarks No other than pollen and pollen from the greenhouse is se
27. ate room for the stocking of recommended required required required clean cages feed and caging material 14 Surfaces resistant to decontaminants required required required required impermeable and easy to clean cages benches cages benches cages benches cages benches floor floor walls ceiling floor walls ceiling 15 Facility for the washing of cages required required required required 16 Automous electricity backup system not require not required recommended required 17 Fire alarm not taking into account not required not required required required local fire regulations 18 System that enables communication not require not required required required non with the outside world manual operation VENTILATION 19 Input airflowsystem separated from not require not required recommended required adjacent laboratories 20 Extract airflowsystem separated from not require optional recommended required adjacent laboratories 21 Input and extract airflowsystem not require optional required required interconnected to prevent accidental overpressure p 22 Input and extract airflowsystem can be not require optional required required n E closed using valves bu 50 Biosafety in the laboratory Specifications Containment level Al A2 A3 A4 23 Negative airpressure in the not required optional required control and required control and controlled zone when compared alarm systems alarm systems
28. atory to contain its own equipment not required not required required required 34 Protective clothing required required required required specific for the specific for the containment zone containment zone 35 Decontamination of the protective not required not required recommended required clothing before it leaves the containment zone 36 Gloves not required optional required required 37 Mouth mask not required not required optional required 38 Face protection eyes mucosa not required optional required required 39 Fysical containment of live recommended required required required micro organisms closed systems 40 Generation of aerosols minimize minimize prevent prevent 41 Specific measures to prevent the spread not required recommended required required of aerosols including appropriate equipment 42 Mechanical pipetting required required required required 43 No drinking eating or smoking no use require required required required of cosmetics no manipulation of contacts or the stocking of consumables 44 Appropriate laboratory registers required required required required 45 Verification of control measures and required required required required protective equipment 46 Notice on the use of decontaminants require required required required 47 Decontaminants in the siphons not required not required recommended required 48 Instruction of the personnel require required required required 49 Written instructio
29. bts about the possibly hazardous characteristics of the material used i e non tested human blood The people responsible for biosafety and or workers pro tection should be notified and if necessary a doctor and insurance company with a view to recovering the costs of expensive drugs as well Immediately after the accident the f Contamination accidents decontamination and inactivation 29 appropriate measures should be taken Antiviral drugs for instance should be administe red to the victim as soon as possible after the accident Prid An accidental prick or cutting injury or a splash in the eye or on the mucosae with mate rial that is infectious to humans should be dealt with as follows 1 allow the wound to bleed freely 2 rinse the wound thoroughly 3 decontaminate the wound 4 provide further care bandages etc if necessary 5 make a note in the first aid notebook Depending on the type of agent the victim has been exposed to further measures may be necessary When the victim has been exposed to non tested human blood for instance it should be determined whether he she has been infected with Hepatitis B C or HIV and if so appropriate therapy should be decided on How to decontaminate materials and work surfaces z lt iu 30 There are several disinfectants available all of which are characterised by their own specific mode of action In general there are two common modes of action
30. c A2 containment measures will not be enough to prevent the unwanted spread of the virus The mouse will have to be kept in special cages fitted with a filtertop and all the waste faeces urine bedding material etc will have to be inactivated If the same experiment would be done in a pig then again additional measures would be necessary but measures that are different than in the case of the mouse It will not be possible to keep the pig in a filtertop cage The stable as a whole will have to provide the proper con tainment This means that there will have to be an airlock negative airpressure fumigation will have to be possible personnel will have to wear special protective clothing that will remain in the airlock when leaving the stable and there will have to be special equipment for the collection and inactivation of fae ces and urine a closed collection system connected to a inactivation tank Guidelines for the classification of GMO activities Lose hee 63 THE RISK GROUPS OF SOME RELEVANT In this annex the risk groups are given of a number of relevant pathogens This list is a shortened version of the lists given in the annexes of Vlarem Il If a particular pathogen is not present on this shortlist the complete Vlarem list should be consulted Is the pathogen also not present on the Vlarem list then you should contact your internal or external biosafety expert or the Section Biosafety and Biotechnology of the WIV w
31. cal r mal Waste from animal houses belongs to the same categories mentioned above Transgenic animals such as mice or rats should be killed and taken to an incinerator The bedding material and faeces of these animals should not be regarded as biological waste This material can be disposed of as residual waste without inactivating it beforehand It is a different matter however when tests involving genetically modified and or pathogenic micro organisms are performed on animals The micro organisms used are a source of contamination Animals containing these micro organisms should therefore be killed and sterilised or incinerated as hazardous medical waste and all the bedding material and faeces should be collected and inactivated either in house or in an incinerator Waste from plants wA r Me 34 Care should be taken in dealing with waste material from transgenic plants The plants reproductive parts should all be collected Flowers containing pollen seeds and depen ding on the type of plant other reproductive parts should be collected and inactivated see chapter four of this booklet There are different ways to inactivate this material sterilisation is the most rigorous method but simpler methods may sometimes be enough like chopping the material into small pieces Also steaming of the plant material or soil that has been into contact with the transgenic material is a very good means of inactivation All non repro
32. chlorine deteriorate quickly They should be replaced at least once a week to ensure their decontaminating effect Formaldehyde Formaldehyde is a gas that is effective against all micro organisms provided that it is used at a temperature higher than 20 C and at a humidity of at least 70 It is suited for the decontamination of fluids safety cabinets and even whole laboratories by means of atomisation Note that formaldehyde is a suspected carcinogen Moreover it is an irritant and should not be inhaled For this reason formaldehyde is only used in exceptional cases when a safety cabinet or the whole laboratory needs to be deconta minated Examples are an airtight L3 laboratory that has been contaminated or an air tight animal house the floor and walls of which may be covered with dangerous micro organisms Decontamination by means of formaldehyde is a time consuming and dan gerous procedure that can only be performed safely by specialists It is therefore recom mended that the safety and prevention service be consulted before such procedures are carried out Hydrogen peroxide Like hypochlorite this substance is effective against all types of micro organisms owing to its oxidising characteristics It is used as a 6 solution Hydrogen peroxide is not sui ted for use on aluminium copper sink or bronze Modern wide spectrum disinfectants There are many modern wide spectrum disinfectants on the market Mostly they combine differen
33. containment measures In case the preceding step does not give you a decisive answer you should subsequently 1 look up any missing information that might help you in assessing the materials 2 consult your superior 3 consult your group leader 4 consult internal biosafety experts see annex 5 to this booklet or external biosafety experts www biosafety be Working with non pathogenic genetically modified yeasts Classification Non pathogenic genetically modified yeasts such as Saccharomyces cerevisiae Schizosaccharomyces pombe and Pichia pastoris can all be handled under L1 contain ment conditions provided that no hazardous genes e g genes producing toxins have been cloned into the organism The yeast strains used in laboratories are all deficient in some way and need a special feeding medium to which a particular essential substance has been added These deficiencies are often deficiencies in amino acid Laboratory requirements The standard L1 laboratory facilities as required for E coli are sufficient here However it is recommended to perform procedures during which the yeasts are exposed to the envi Biosafety in the laboratory ronment in a safety cabinet Yeasts are airborne and able to survive in the air for long periods of time In this way they can contaminate all kinds of other cultures In addition sensitised people might suffer from allergic reactions after having been exposed to yeast or its spores For t
34. d egg or the modification of plants by means of particle bombardment Depending on the system used the vector will remain present in the final GMO or not Risk assessment GMOs like non GMOs are neither intrinsically hazardous nor intrinsically safe That is why risk assessment is performed on a case by case basis The risk assessment procedure consists of of three subsequent steps 1 Firstly the characteristics of the host vector and donorsequences that are potentially hazardous like pathogenicity toxicity the possibility of uncontrolled spreading of the organism or its genetic material are identified This leads to a preliminary identification of the risk level 2 Secondly the circumstances under which the organisms can be handled safely are deter mined taking into account the following aspects The characteristics of the environment that could be exposed to the GMOs The type and scale of the activity Any non standard activities or actions 3 Finally a risk class is determined based on the results of the first two steps As for pathogens four risk classes have been determined for GMOs Risk classes as defined by the European directive 98 81 EC concerning the contained use of genetically modified micro organisms Biosafety in the laboratory There is already quite some experience with the risk assessment of GMOs In annex 2 of this booklet a set of guidelines is given for the risk categorisation of GMOs The
35. d required a programme to combat rodents and insects 8 Observation window or equivalent not required optional optional required system that enables one to see who is working in the laboratory 9 Decontamination facilities for required sinks required sinks required sinks required sinks and the personnel in the airlock or shower the latter fitted near the exit with a chemical sprinkler in case use is made of isolation suit fitted with positive airpressure 10 Sinks that can be operated not required optional required required non manually 11 Coathooks or changing room for recommended required required required the protective clothing 12 Tubing fitted with a system that not required not required recommended required prevents backward flow of fluids 13 Surfaces able to withstand acids required required required required bases organic solvents and bench bench bench floor bench floor walls decontaminants water resistant and ceiling easy to clean exe s Containment requirements 47 Avg 48 Specifications Containment level ll 12 B l4 14 Autonomous electricity backup system not required not required recommended required 15 Fire alarm not taking into account not required not required required required local fire regulations 16 System that enables communication not required not required required required non
36. ditional containment measures that might be necessary 1 The formation and dissemination of aerosols cannot be minimised cfr variation above Using a class Il safety cabinet will be required in many cases 2 GMOS or pathogens are handled for a longer period of time during a non destructive measure ment without any containment measures being taken If the organisms pose a hazard it should be determined whether they might spread If so the laboratory worker and the laboratory itself often need extra protection for instance negative air pressure and HEPA filtration of the exhaust air and or gloves and face protection 3 GMOs or pathogens are manipulated in fermenters and purified in a downstream process Some fermenters are more leaktight than others which is also true of downstream processing equip ment When risk class Il IIl or IV organisms are used the fermenter s air exhaust system should contain a special hydrophobic absolute filter or a HEPA filter 4 GMOs or pathogens are administered to a plant or animal internally or externally It should be determined on a case by case basis whether dissemination of the GMO or pathogen to the environment should be countered and how The measures to be taken strongly differ from one plant or animal to another The method of administration is often decisive for the way in which a micro organism is able to spread What should one do when there are doubts about the risk class and necessary
37. ductive parts of transgenic plants can be composted dry or wet and after being composted this material can be disposed of as residual waste or garden waste When plants have been infected with genetically modified and or pathogenic micro orga nisms they should be inactivated as a whole as well as the soil or other possibly contami nated material Biosafety in the laboratory 7 WORKING WITH COMMONLY USED LABORATORY Classification Genetically modified E coli K12 B and C strains and all fages can be manipulated in labo ratories that meet the L1 criteria on condition that the vectors used are not self transmissible the genes inserted do not produce hazardous products such as toxins or have hazar dous effects These K12 B and C strains are attenuated weakened laboratory strains that have been biologically constrained and are no longer pathogenic The table below lists some exam ples of classifications which have been made on the basis of the guidelines in annex 2 Because it is assumed that the inserts used in these examples are not hazardous article e or j of the guidelines are applicable depending on whether or not the insert has already been characterised It goes without saying that the nature of the insert is a strong deter minant of the final classification This means that when hazardous inserts are used for instance one that can produce a toxin another article of the guidelines will be applicable
38. e designed to protect the worker in the first place and the environment in the second place A distinction is made between three types of safety cabinets class 1 I and Ill A class 1 cabinet is a fume hood in which exhaust air passes through a HEPA filter This type of cabinet provides wor ker protection but no experiment protection Containment a combination of infrastructure and working practices f gt 21 front opening sash exhaust HEPA exhaust plenum onw gt D Room air B contaminated air 3 HEPA filtered air Class Il safety cabinets A class 11 safety cabinet a downflow cabinet provides worker environmental and experi ment protection These cabinets have a downward laminar airflow There are many diffe rent types of class ll cabinets front opening sash exhaust HEPA filter supply HEPA filter rear plenum blower m g G gt D Room air B contaminated air 3 HEPA filtered air The proper use of a class Il safety cabinet A class Il safety cabinet can only provide effective worker experiment and environmental protection if it is properly used For this reason disruptions of the air flow inside the cabi E net should be avoided as much as possible which boils down to the following M 22 Biosafety in the laboratory Guidelines for the proper use of a class Il safety cabinet 1 Prepare an experiment thoroughly and collect all necessary materials bef
39. ectant wash your hands afterwards Minimise the creation and spread of aerosols by only using closed tubes to centrifugate preventing caps from becoming wet heating wet inoculation needles in the proper way i e first heat the shaft then the eye allowing inoculation needles to cool down before putting them back into the fluid never using force to empty pipettes but allowing gravity to do its work pouring out fluids in a gentle manner and never from a great height Mouth pipetting is forbidden always use a pipetting bulb or other mechanical pipette use a mechanical micro pipette for small amounts Decontaminate used materials before washing and reusing them this can be done by autoclaving them or by immersing them in a vali dated disinfectant Inactivate biological waste e put the waste in a waste basket for biological waste The waste should then be transported to the autoclave or to a waste incinerator that is capable of burning contaminated hospital waste Wash your hands after the experiment and before leaving the laboratory use disinfecting soap to do so Biosafety in the laboratory In addition to following these safe working practices it is important to 1 keep a detailed laboratory notebook containing a daily account of all biological mate tials used and all manipulations performed 2 routinely check the biological material you work with If you receive material from a colleague you shou
40. eds unlikely especially when windows are fitted with insect nets Wheat Self pollinator The spread of No special remarks The spread of roots and pollen is very unlikely to have stembases should also be an impact prevented Transgenic animals The unwanted spread of transgenic animals should be prevented Depending on the ani mal this can be very easy or rather difficult Small rodents like mice should be kept in appropriate cages and the animal houses should be designed in such a way that it is impossible for the animals to escape When a genetically modified micro organism or a wild type pathogen is administered to the animal it should be determined on a case by case basis how to prevent the micro organism from spreading It may be necessary to keep the animals in individually ventilated cages and to inactivate all materials that have been in contact with the animals for instance the bedding material When cells or other biolo gical material are used in animals it should be taken into account that viruses may be present in this material Some cell lines are contaminated by viruses If such viruses are present the containment measures should be adapted if there is a risk that the virus might spread The spread of organisms in the laboratory f AE Mn a z lt iu 20 5 CONTAINMENT A COMBINATION OF INFRASTRUCTURE AND WORKING PRACTICES Four levels of containment appropriate to the four risk classes for path
41. ee clarification of terms Biosafety in the laboratory 3 CLASSIFICATION AND VIEN Pathogenic organisms Organisms are divided into four categories of risk Organisms that are not able to cause disease belong to risk group 1 Pathogenic organisms belong to the risk groups 2 3 or 4 depending on their degree of pathogenicity and the availability of effective treatment To distinguish between the classification of natural non modified pathogens and GMOs the pathogen classification uses the term risk groups or sometimes also biological risk class while for the GMO classification the term risk class is used Below an overview is given of the definitions of the different risk groups Group 1 Very unlikely to cause disease in humans animals or plants Group 2 Human pathogens _ micro organisms that can cause disease in humans and pose a hazard to persons that are directly exposed to it Their spread to the commu nity is unlikely Profylaxis or effective treatment is mostly available Animal pathogens micro Jorganisms that can cause disease in animals and that possess in different extend one of the following properties limited geographi cal importance transmission to other limited or non existent species absence of vectors or carriers Limited economic and or medical impact Profylaxis and or effective treatment is mostly available Fytopathogens micro Jorganisms that can cause disease in plants but for which there is no high
42. eful comments to earlier edi tions of this booklet Ann Van Gysel communication manager VIB Bernadette Van Vaerenbergh WIV Section Biosafety and Biotechnology Greet Van Eetvelde environmental coordinator University of Ghent Huub Schellekens platform committee VIB University of Utrecht Kim De Rijck Mieke Van Lijsebettens VIB department of Plant Genetics University of Ghent Hilde Revets VIB department of Molecular and Cellular Interactions Free University of Brussels Peter Brouckaert VIB department Molecular Biomedical Research University of Ghent Thierry Vandendriessche VIB department Transgene Technology and Gene Therapy Catholic University of Leuven Wim van de Ven VIB department Human Genetics Catholic University of Leuven Hubert Backhovens VIB department Molecular Genetics University of Antwerp Aknowledgement The list below is meant to serve as an overview of the persons responsible for the safety in biotechnolo gical laboratories These persons differ from one department to another Fill out the list below and make sure that you have it within reach in your laboratory Telephone Other Emergence number Laboratory responsible person First aid responsible person Location of first aid kit Biosafety coordinator Workers protection contact Company doctor Waste contact person Regulatory affairs manager VIB Ren Custers 09 244 6611 rene custers vib be
43. ences for its spreading 46 Clarrification of terms In this annex the belgian containment requirements are given for laboratories animalaria and greenhou ses The requirements for hospital rooms and large scale processing equipment are not given here These requirements can be found at www biosafety be Also the requirements for so called quarantine orga nisms are not given here If you want to work with one of the pathogenic organisms that are present on the lists of quarantine organisms of the FAVV one should ask them for the requirements to work with these organisms Belgian requirements for L1 L4 laboratories for work with icall lified y 1 Equipment and technical requirements 1 The laboratory is separated from other not required not required required required working zones in the same building or is located in a separate building 2 Entry to the laboratory via an airlock not required not required equired or only required entrance via L2 3 Doors can be locked not required required required required 4 Doors that close automatically not required optional required required 5 Windows that cannot be opened not required not required required required but closed during able to withstand experimentation breaking 6 Airtight laboratory that enables not required not required required required decontanimation with a gas fumigation 7 Laboratory furniture that enables not required recommended require
44. er risk of an epidemic when they are accidentally dissemina ted into the Belgian environment Profylaxis or effective treatment is available Non indiginous or exotic fytopathogens that are not able to survive in Belgium because of the absence of target plants or because of unfavourable weather conditions belong to this risk group Group 3 Human pathogens micro organisms that can cause serious disease in humans and pose a hazard to persons that are directly exposed to it There is a risk of spread to the community Profylaxis or effective treatment is mostly available Animal pathogens micro organisms that can cause serious disease or epizotic in ani mals Spread to other species is more than possible Some of these pathogenic agents require specific sanitary measures Profylaxis or effective treatment is mostly available Fytopathogens micro Jorganisms that can cause a disease in plants with important economic or environmental consequences and for which treatments are non existent difficult to apply or costly Accidental spread may lead to local epidemics Exotic strains of fytopathogens usually occu ring in the Belgian environment and not in the list of the quarantine organisms also belong to this risk group M Classification and risk assessment 9 Belgian definitions of the different risk groups Most definitions of risk groups are developed from the WHO classification of infective micro organisms In addition to classifying
45. erforming small scale standard laboratory procedures only Step 1 Is the combination of host and vector suited for risk class 1 To find this out the E coli strain should be looked at first all E coli K12 B and C strains are suited for risk class 1 and it is Biosafety in the laboratory therefore sufficient to check in available catalogues with suppliers or in strain databases ATCC DSMZ etc whether the JM109 strains belong to these classes of strains The answer in this case is yes JM109 belongs to the E coli K12 class Next the vector s suitability for risk class 1 should be determined PUC18 is a well characterised vec tor that has no hazardous properties and is non self transmissible The vector is suited for risk class I a detailed description of the requirements for vectors can be found in annex 2 chapter A to this booklet A description of what is meant by well characterised is also given there It can therefore be concluded that the combination of this specific host and vector is suited for risk class I Step 2 Now the relevant containment category can be looked up in annex 2 In this particular case category 1 of the first direction of annex 2 chapter A is applicable since the combination of host and vector is suited for risk class 1 However a final assessment cannot be given before the risk the insert itself poses has been determined Step 3 In this case a piece of bovine genetic material encoding
46. es and working practices that need to be applied The number of viral vector systems composed of components originating from different viruses is growing rapidly The most well known example is the pseudo typing of retrovir al vectors using the VSV G protein Such combinations of viral components deserve special attention since the host range may change for instance from non infectious for humans to infectious for humans The risk class should be adjusted to the actual risk Biosafety in the laboratory Classification Most researchers work with disarmed A tumefaciens strains which means that the strains are no longer oncogenic They no longer cause crown galls These disarmed strains belong to risk class 1 This does not apply to wild type strains or A rhizogenes Strain vectors Classification Classification Comments rule on the assumption that non hazardous inserts are used as applied in the schemes of annex 2 Laboratory requirements Basic L1 containment measures are sufficient for disarmed A tumefaciens strains It is enough to follow the rules laid down for E coli K12 As far as not disarmed tumefaciens strains or A rhizogenes are concerned basic L2 containment measures will suffise In cases when the creation and dissemination of aerosols containing the bacterium cannot be avoided a class Il safety cabinet should be used Waste All materials whether solid or fluid should be decontaminated as was the
47. ese guidelines In chapter A there is a description of how to use the classification schemes Also the necessary definitions of T1 T2 and T3 toxins are given as well as the requirements for risk class 1 hostorganisms and vectors For activities with transgenic plants chapter B gives some guidance Activities with transgenic animals and especially the combination of the use of genetically modified micro organisms or cells with animals are difficult to classify Chapter C gives some guidance for these activities Of course there are cases that are not described in these guidelines In such cases it is best to contact internal or external biosafety experts as given in annex 5 in this booklet Sources These guidelines have been developed from the guidelines from the dutch genetic modification advisory committee COGEM as far as applicable in Belgium and from the classification guidelines in the annexes of section 51 of Vlarem Il CHAPTER A GUIDELINES FOR THE CLASSIFICATION OF ACTIVITIES INVOLVING GENETICALLY MODIFIED MICRO ORGANISMS AND CELLS Classifica i On the following pages classification schemes are presented that can be used to determine a first indica tion of the necessary containment level As already explained in chapter 3 and in the example in chapter 7 of this booklet this is only a first but important step To come to a final conclusion one still has to consider 1 the environment to which the GMO would be exposed 2 the type a
48. etically closely related species which can exchange genetic material by natural physiological processes where the resulting micro organism is unlikely to cause disease to humans animals or plants Self cloning may include the use of recombinant vectors with an extended history of safe use in the particular microorganisms R t DN A GMO Today a whole range of organisms can already be genetically modified a o bacteria yeasts fungi insects fruit fly parasites nematodes plants frogs mammals mice rats rabbits goats sheep pigs cattle Genetic modification in general involves the following components Classification and risk assessment x nt wk x 11 A host organism the organism which is to be modified note that the meaning of the term host in this context differs from that in the context of pathogenic organisms See clarification of terms A donor sequence or insert isolated from a certain organism the donor organism However synthetically produced DNA sequences are also being used more and more often These sequences can be identical to sequences present in living organisms but they can also be completely new And in many but not all cases a genetic vector In the case of transformation of bacteria plasmids are mostly used as a vector In other cases viruses or viral vectors may be used Examples where no genetic vector is used are the micro injection of DNA in the pronucleus of a fertilise
49. eusing it or discarding it as waste Reproductive plant parts should be inactivated before they are disposed of as residual waste Biosafety in the laboratory 9 REPFEREN Begrip voor Veilige Microbiologische techniek theoretisch inleiding voor de praktische cursus Commissie ad hoc recombinant DBA werkzaamheden 1987 Veilig werken met micro organismen en cellen in laboratoria en werkruimten theorie en praktijk concept oktober 1997 Nederlandse vereniging voor Microbiologie Leidraad voor een Handboek GGO ingeperkt gebruik BVF platform 2001 Laboratory Biosafety Manual second edition WHO Geneva 1993 Directive 90 219 EEC as amended by directive 98 81 EC Vlarem 1 and ll section 51 Biosafety cabinets CDC Atlanta USA http www biosafety be http www hc sc qc ca hpb Icdc biosafety msds index html http www ebsa be http www cde gov od ohs References 45 JON Aerosol very small droplets of fluid that can spread via air Amphotropic able to infect both mouse and human cells Biological agent in its legal definition this means organism that is able to cause disease A Donorsequence a piece of genetic material that is transferred from one organism the donor to the receiving organism also named insert In many cases the insert nowadays is made synthetically Ecotropic only able to infect mouse cells Host 1 in the context of natural pathogens this means the organism that
50. ganisms that are able to spread through the air very small droplets play a role but infection may also be the result of direct contact for instance with hands handkerchieves or clothes droplets dust EN secondary reservoirs handkerchieves cl othies ER Figure 2 the spread of micro organisms via air 14 Biosafety in the laboratory Routes of contamination Laboratory personnel may be exposed to organisms in different ways Any open source of organisms for instance an open petri dish may lead to the spread of organisms However under normal circumstances a container holding living pathogens of GMOs will only be opened in semi sterile surroundings so as to prevent contamination of the container s content itself for instance close to a Bunsen burner or in a safety cabinet In practice the cause of most laboratory infections is unknown When the cause of the infection is known it often concerns prick accidents spilling broken glassware mouth pipetting or biting or scratching by a laboratory animal Aerosols One of the routes of infection that deserves special attention is infection through aerosols Aerosols are very small droplets of fluid that can spread through the air They are formed during activities such as opening bottles containing fluids and having a wet cap vor texing blending emptying a pipette by blowing or heating a wet inoculation needle in a flame The formation of aerosols should be avoided
51. handling non modified micro organisms or cells see chapter 5 Risk class 2 3 and 4 agents may only be handled in laboratories that are specially designed for that purpose ee eee hs isms GMOs Recombinant DNA technology has become so important that one can no longer imagine modern biological and biomedical laboratories without the technique Escherichia coli K12 is the number one laboratory organism which is used by almost every researcher as a means of cloning or expressing genes or sequences Biosafety in the laboratory The following GMOs are excluded from the regulations and also from the scope of this booklet if they have been made using the techniques listed below on the condition that they do not involve the use of recombinant nucleic acid molecules or GMOs other than those produced by one or more of the techniques listed below Mutagenesis Cell fusion including protoplast fusion of prokaryotic species that exchange genetic material by known physiological processes Cell fusion including protoplast fusion of cells of any eukaryotic species including production of hybridomas and plant cell fusions Self cloning consisting in the removal of nucleic acid sequences from a cell of an orga nism which may or may not be followed by reinsertion of all or part of that nucleic acid or a synthetic equivalent with or without prior enzymatic or mechanical steps into cells of the same species or into cells of phylogen
52. have been brought into the host can give rise to the formation of autonomously replicating virusparticles h The sequence contains genetic information Risk group 4 for the formation of a defect for eucaryotic Risk group 3 cells infectious virus of respectively risk Risk group 2 group 4 3 or 2 and the viral sequences that havebeen brought into the host cannot give rise to the formation of autonomously replicating virusparticles i The sequence contains genetic information hat codes for a hazardous gene product other than in f j The sequence does not contain genetic infor mation that codes for a hazardous gene product 10 Activities in which characteri sed genetic material is or has been added Gui material is or has been added that has not been 1 Activities in which genetic characterised 1 13 L2 L2 L4 fs 12 12 L2 L2 L2 L2 L2 L2 L3 L2 L2 L4 L3 L2 12 L2 L2 L2 L2 delines for the classification of GMO activities ak derd node des 62 CHAPTER B GUIDELINES FOR THE CLASSIFICATION OF ACTIVITIES IN GROWTH CHAMBER AND GREENHOUSES INVOLVING TRANSGENIC PLANTS OR PLANTS THAT CARRY GENETICALLY MODIFIED MICRO ORGANISMS Containment level G1 Sterile or sterilised plants Strict autogamous plants strict selfpollinators
53. his reason yeast is often manipulated in a separate laboratory Waste All waste consisting of or containing genetically modified yeasts should be inactivated or decontaminated Note As was the case for E coli the organism will move one level up in both risk class and the corresponding measures to be taken when a gene that produces a hazardous substance such as a toxin is cloned into the yeast Working wit cally modified cell I Classification Classifying animal or human cell lines is not that simple The determining factor is the cells ability to produce hazardous biological agents notably viruses First of all a distinc tion should be made between primary cells and established cell lines Primary cells should at least be classified as L2 organisms When established cell lines are involved it should be determined whether they might produce virus particles taking into account any viral sequences that might be present in the vector being used in combination with the cell line Some examples Viral Vector by way Classification Classification Comments sequences of example only in cell line Working with commonly used laboratory organisms on the assumption that non hazardous inserts are used hazardous inserts would require a higher risk class as applied in the schemes of annex 2 Laboratory requirements The laboratory requirements may vary depending on the classification A basic L1 labora
54. ilise or not mobilisable at all Donor The plasmid contains sex pili chromosome genes that code for the Single stand DNA is sex pili transferred through proteins F Plasmid the pilus to the receiver Receiver Fig 4 Transmission of a self transmissible plasmid through conjugation Biosafety in the laboratory Animal and human cells Animal and human cells cannot spread to the environment just like that In addition non contaminated cells are unable to spread genetic material to the environment by accident Animal and human cells cannot survive in non sterile surroundings Cells that are specially designed to survive in non sterile surroundings such as fish or frog eggs are an exception to this rule As regards non contaminated cells the measures that are taken to prevent the cell culture from being contaminated are sufficient to prevent the cells from being spread to the environment Genetic material of animal or human origin can only be spread to the environment when the cells involved are infected by biological agents such as viruses that are able to mobilize their genetic material From a biosafety point of view the question whether or not cells are infected by biological agents is very important Any viruses pre sent may represent a danger to the researcher or to the environment and any safety measures should take account of this Viruses A distinction can be made between wild type viruses and viral vectors
55. iment Which of the following decontaminants does not work against spores 1 chlorine 2 formaldehyde or 3 ethanol True or false contaminated materials that will be reused only have to be washed They do not have to be decontaminated What should one do after genetically modified bacteria have been spilled on the labora tory bench Mention five important routines for working in a class ll safety cabinet Biosafety in the laboratory SELFTEST ANSWERS uoypununjuorap sof ubis yprtoppur 10160101Q pasn opp owvu boy jauiqvo Gafos ays mo yf OL saunu sayjoun sof Burysom jaurgo ay ava 6 1uowtuadra ay 4 1fD spisum ay pup wouaq ay ajpunupjuooop 8 mojfum or fo oouwqansip osuuiunu o Ayuab aow 4 Jauiqns ay ui ybnous daap suom 9 spijsum yovq puo Juo4f aij uo puagout Kuv and jou op G upssodau J314S 24D JOY j2uiqvo Y ui s puajvut 2i Ind Quo p q40m Buus ofoq SPYSA IY PUD jaurgo ay UL 1 2uaq IYI 3jpunuvjuooop c q4om Duivjs auofag sajnu OT uo jauiquo y vang z NOK puryaq 401D40qn ZT 211 fo 400p y 3s0 9 1 34p saunos JuDpoduy IUMO YI 40 JOUDYJa 32uvjsut AOL JUDUILUDJUOIap v UL p qDos SANSSIJ ynn ybnosoys paypuuupjuosap aq pjnoys yous aij puy Asom payouiunjuos D31Do 01q 4of 12 SDQ ASON IY OJUL UMOLY PUD s nss1 40 SIMOJ 42dvd una padowas aq Pinoys vuajovq PAAS aug payspm aa Kay a4ofag Supa aayoaffa 421 jouv 40 uOIDS 1131S q pajmununpjuooap aq 0 paau pasnaa aq jun J
56. indicates that work is being done with biologically hazardous organisms starting from risk class 2 Introduction f n AD What is biosafety The biosafety of laboratory work is the central focal point of this booklet Biosafety is about the intrinsic hazards of living organisms and how to handle them safely Genetic material as such naked DNA can be dangerous as well Before starting to work with pathogens or genetically modified organisms GMOs in a laboratory one should stop and think about the possible hazards of these organisms and take proportionate mea sures to minimise any risks for human health and the environment Safety first What are the hazards Biological material and living organisms are neither intrinsically dangerous nor intrin sically safe Any danger will depend on the characteristics of the material or the orga nisms Characteristics that represent a danger are the following e Pathogenicity The pathogenicity of an organism indicates whether an organism for instance a bacte rium a virus fungus or a parasite is able to cause a disease in a plant animal or human Factors like infectious dose virulence and the production of toxins by the pathogen play a role in the extent to which the organism is able to cause disease Infectious dose Contamination by one pathogen does not automatically lead to infec tion The infectious dose differs from one pathogen to another Below some exa
57. ine herpesvirus 1 Bovine herpesvirus 2 Bovine herpesvirus 3 Bovine herpesvirus 4 Chimpanzee herpesvirus pongine herpesvirus 1 Cytomegalovirus Human herpesvirus 5 Cytomegaloviruses of mouse guinea pig and rat Epstein Bar virus EBV Human herpesvirus 4 Herpes virus B Orthomyxoviridae Avian influenza virus A Fowl plague Equine influenza virus 1 H7N7 and 2 H3N8 Influenza viruses Types A B amp C Papovaviridae Animal papillomaviruses Human papillomaviruses HPV Bovine polyomavirus BPoV Monkey SV40 SA 12 STMV LPV Paramyxoviridae Measles virus Mumps virus Parainfluenza viruses types 1 4 Parvoviridae Adeno associated viruses AAV Canine parvovirus CPV Human parvovirus B 19 Picornaviridae Coxsackieviruses Polioviruses Swine vesicular disease virus Bovine rhinoviruses types 1 3 Human rhinoviruses Poxviridae Entomopoxviruses Fowlpox virus Other avipoxviruses Camelpox virus Cowpox virus Horsepox virus Monkeypox virus Rabbitpox virus variant of vaccinia Vaccinia virus Variola major amp minor virus White pox Variola virus Swinepox virus Reoviridae ortho reoviruses Human rotaviruses Mouse rotaviruses EDIM epizootic diarrhoea of infant mice Rat rotavirus Retroviridae Avian leucosis viruses ALV Avian sarcoma viruses Rous sarcoma virus RSV Bovine foamy virus Bovine immunodeficiency virus BIV Equine infectious anemia virus Feline immunodeficiency virus FIV
58. is naturally infected by the pathogen 2 in the context of genetic modification this is the organism receiving the genetic modi fication gt HEPA High Efficiency Particulate Air filter s Pathogenicity ability to cause disease Reproductive able to grow out to a autonomously functioning organism Toxicity poisoning Transduction equals viral infection However this terms is mostly used for infection of bacteria by fages and infection of cell by a viral vector _ Transformation 1 in the context of tumours this means the alteration of a normal cell into a infinitively dividing tumour cell 2 in genetic modification the term is generally used for the process of genetic modifica tion transformation genetic modification Vector literally this means carrier 1 in the context of genetic modification this is the plasmid cosmid artificial chromoso me or other genetic element that is used to carry the genetic modification into the host organism 2 in the context of viral infection of plants the term vector is used for the organisms that carries the virusinfection from one plant to the other In some cases aphids are the vector Viral vector a viral construction developed from a wildtype virus and that can carry a piece of genetic material and can behave autonomously in the case it possesses all the sequences necessary to spread from one cell to the other or defectively if it lacks one or more sequ
59. l required 41 Face protection eyes mucosa not required optional optional required 42 Generation of aerosols minimise minimise prevent prevent 43 Specific measures to prevent the spread not required recommended required required of aerosols including appropriate equipment 44 Mechanical pipetting require required required required 45 No drinking eating or smoking no use require required required required of cosmetics no manipulation of contacts or the stocking of consumables 46 Register in which all activities are notified require required required require transport of laboratory animals inoculation of GMM s etc 47 Verification of control measures and require required required required protective equipment 48 Notice on the use of decontaminants require required required required 49 Decontaminants in the siphons not required recommended required required 50 Instruction of the personnel require required required required 51 Written instructions on the biosafety required required required required procedures 52 Efficient vector control eg for rodents recommended required required required and insects 53 Isolation of laboratory animals used required required required required separate room not required separate room recommended period to be specified in the authorisation separate room required period to be specified in the authorisation WASTE MANAGEMENT 55 Inactivation of biologic
60. l safety cabinet should be used since a horizontal laminar flow cabinet does not provide sufficient worker and environment protection Access to the laboratory is subject to strict rules Waste All solid fluid and contaminated materials should be inactivated or decontaminated before they are reused or discarded as residual waste Sterilisation is the recommended method to this end Alternatively the material can be transported in a special container for infectious medical waste to an incinerator suited for the incineration of hospital waste Note Activities involving different types of viral vectors should not be performed in the same laboratory at the same time let alone in the same safety cabinet After finishing work with a particular type of virus the researcher should clean and decontaminate the work surface and the safety cabinet before conducting a new experiment involving another type of virus In the above it has been assumed that the inserts used were non hazardous When viral vectors are used especially vectors that insert their genetic material in the cell s genome extra care should be taken when for instance dominant cellular oncogenes are used The insert will then be classified in a higher risk class Genes encoding some immune modula ting proteins can also be regarded as hazardous In some cases a risk class Ill classification will be necessary for which a lot of effort is required due to the stringent containment measur
61. ld always check whether it is indeed the material you expected to receive check the strain the plasmid and its restriction pattern It is equally important to check your own strains on a regular basis Alternatively you can start from a new clean stock on a regular basis Any basic laboratory facility where the safe microbiological techniques are applied meets the requirements of containment level 1 The laboratory itself does not need to be equip ped with more than the basic facilities such as a floor wall ceiling smooth readily cle anable work surfaces and a sink Containment levels 2 3 and 4 require a number of addi tional precautions a o gloves should be worn when experiments are being conducted access to the laboratory is restricted to authorised personnel only an insect and rodent control programme is in effect all liquid effluents released from sinks and showers should be inactivated additional measures to prevent the creation of aerosols should be taken for instance procedures with a potential for creating aerosols should be conducted within a safety cabinet clothing should be decontaminated before leaving the facility etc An overview of the requirements of containment levels 1 to 4 is given in annex 1 The use of gloves There are many misunderstandings regarding the use of gloves and in many cases gloves provide a false sense of safety For level 1 activities gloves are not required The app
62. lication of the safe microbiological techniques is sufficient to work safely Gloves are no basic requirement for level 2 either But they should be worn when direct contact with the bio logical material poses a hazard or prick or cutting injuries might occur for instance when people are working with sharps and biologically hazardous material Gloves are mostly used to prevent contamination of the experiment with micro organisms DNA or RNA pre sent on your hands One should not forget however that gloves can bear this material just as well especially when they have been worn for a longer period of time and when they have been in contact with all kinds of materials Thus when using gloves laboratory staff should keep the following guidelines in mind Containment a combination of infrastructure and working practices s AT 27 Only wear gloves when strictly necessary i e only when there is a genuine risk that you or the experiment might be contaminated Only wear gloves when you are actually conducting the experiment Do not wear gloves when handling all kinds of other materials like closed buckets or when writing in your laboratory notebook When using gloves change them on a very regular basis Restricted access W 28 From level 2 activities onwards the access to the laboratory is legally restricted This means that only personnel that meets specific entry requirements is authorised to enter the labo ratory
63. lture flasks pipettes etc e materials that are not necessarily contaminated but cannot be thrown into an ordinary waste disposal bag because they have sharp edges or look dirty bones blood etc Biological waste does not include radioactive contaminated material Such material should be dealt with separately How to manage biological waste A distinction can be made between solid or pasty waste and fluid waste Depending on whether the material is inactivated and or chemically polluted waste is categorised as fol lows Contamination accidents decontamination and inactivation f iu 33 A Type of waste Inactivated Chemically polluted Hazardous medical waste is collected in special containers that are suited for the transport of medically hazardous waste Once closed these containers cannot be opened anymore In addition they are resistant to leaking so that falling from heights poses no problem These special containers should be transported by an accredited transporter to an incinera tor that is suited for the incineration of medical waste on a very regular basis The follo wing guidelines exist for the temporary stocking of the containers and the transport to the incinerator a maximum of 2 months at a temperature of no more than 4 C a maximum of 2 weeks at a temperature of no more than 20 C a maximum of 1 week if the temperature of the stock room can become higher than 20 C Biolnai
64. micro organisms or their remains may accumulate in the laboratory and thus become a potential source of conta mination for the work of other laboratory staff It goes without saying that accidents with organisms posing a genuine threat either to the laboratory worker and other humans or to the environment risk class 2 3 and 4 pathogens or GMOs are possible as well If such an accident occurs it is an absolute must to clean and decontaminate immediately Spills on damaged skin or spills on intact skin of for instance human pathogenic viral particles should never be disregarded LLL Prevention Since prevention is always better than cure it is important to avoid accidents whenever possible This can only be achieved by working carefully and thoughtfully Experiments should be carefully planned all necessary materials should have been collected before hand and afterwards everything should be put back where it belongs Sloppiness is the major cause of accidents Scalpels for instance should never be left unattended on one s bench Put them in a safe container instead As for needles they should never be recapped after use Place them in a special needle waste container Natica When a laboratory staff member has been exposed to dangerous human pathogens this should always be reported and not only when blood is pouring everywhere An accident should be reported even when there has been only a minor risk of contamination or when there are dou
65. mples are given of human pathogens infectious dose when they are spread by their normal route of infection Pathogen Infectious dose Adenovirus gt 150 pfu intranasaly Influenza virus 790 pfu nasopharyncheal Tuberculosis bacteria 10 bacteria inhalation Thyphoid bacteria 10 bacteria ingestion Pfu plaque forming units Source Material Safety Data Sheets Health Canada Toxicity Toxicity means poisoning Most substances are not poisonous when they are used under normal circumstances The toxicity of a substance is mostly given as an LD50 for verte 6 Biosafety in the laboratory brates in weight units per kilogram body weigth The LD50 LD stands for lethal dose is the amount at which exposure to the substance leads to the death of 50 of the ani mals exposed When the toxicity of living organisms especially bacteria is considered toxicity often coincides with pathogenicity Allergenicity Allergenicity is a non toxic immune system mediated undesired reaction of the body to a substance or agent Immune globuline E IgE and mast cells immune system cells that among other things produce heparin often play a role in the allergic reaction An allergic reaction may lead to sneezing skin irritation asthma attacks chronic lung dis orders and sometimes even to a lifethreatening shock Disturbance of ecological balances The aspect of disturbance of ecological balances is especially relevant for activities involvi
66. n order to achieve containment at the source researchers should if possible Physical Physical containment comprises all physical measures that are taken to protect organisms from the surrounding environment and vice versa This starts with the container holding the organism for instance a tube but it also comprises the structure floor walls ceiling doors and windows of the laboratory as well as benches that are suitable for working with pathogenic organisms or genetically modified micro organisms To allow the handling of micro organisms the benches should have a smooth surface and be readily decontamina ble A sink should be present to allow laboratory staff to wash their hands after an experi ment When a higher containment level is required 2 3 or 4 a number of additional phy sical containment measures will be taken depending on the risk the activity represents such as e class 1 Il or M safety cabinets negative air pressure in the laboratory an air lock a shower HEPA filtration of the exhaust air special cups and other equipment to prevent the spread of aerosols e gas tightness of the laboratory to permit gas decontamination etc An overview of all legally required physical containment measures is listed in annex 1 Safety cabinets A safety cabinet is an important type of physical containment In fact a safety cabinet creates a safe working space within the laboratory Safety cabinets ar
67. nd guidelines exist for GMOs These are designed to protect human health and the environment We want to stress the importance of working safely and responsibly with biological and especially genetically modified material It is the obligation of the researcher to apply all the necessary safety measures It is also the obligation of the researcher to teach all inex perienced personnel the theoretical and practical aspects of biosafety We hope that this booklet together with practical instructions and guidance will prove to be a useful tool to welcome people in a safe manner into the fascinating world of bio technological laboratory research he ces Jo Bury and Rudy Dekeyser General management VIB Preface OAN TINTRODUGTIONS maaa ENNE 5 2 PEBIOSARE iya a NEN MN 6 3 CLASSIFICATION AND RISK ASSESSMENT eee 9 4 THE SPREAD OF ORGANISMS IN THE LABORATORY 14 5 CONTAINMENT A COMBINATION OF INFRASTRUCTURE AND WORKING PRAGT GESE seat ere e MR 20 6 CONTAMINATION ACCIDENTS DECONTAMINATION AND INACTIVATION 29 7 WORKING WITH COMMONLY USED LABORATORY ORGANISMS 35 3 EREFERENGES MENE 45 OMEGCIARRIEIGATIONKOESTERNIS Sn 46 ANNEX 1 CONTAINMENT REQUIREMENTS Wanna nan 47 ANNEX 2 GUIDELINES FOR THE CLASSIFICATION OF GMO ACTIVITIES 56 ANNEX 3 THE RISK GROUPS OF SOME RELEVANT PATHOGENS 64 ANNEX 4 AKNOWEE
68. nd the scale of the activity and 3 possible non standard manipulations The classification scheme is divided into four parts e Part 1 activities with host vectorsystems that do or do not fulfill the criteria for categorisation in risk class 1 and activities with non viral pathogenic hostorganisms e Part 2 activities involving animal cells without viral vectors and with baculoviruses e Part 3 activities involving animal cells in combination with viruses or viral vectors e Part 4 activities in plant cells at 56 Biosafety in the laboratory The schemes should be used as follows In a first step the four schemes are screened in the first direc tion and is determined what type of host and vector is applicable In a second step it is determined in the second direction whether it concerns activities with non characterised or characterised genetic material In a third step it is determined within the applicable type of genetic material characterised or non characterised what type of insert is applicable In the case of non characterised genetic material it is a choice between categories a to e and in the case of characterised genetic material it is a choice between categories f to j From the combination of applicable categories of host vector and insert the applicable classification can be read Definition of d f toxi a A T 3 toxin is a toxin with an LD50 for vertebrates of less than 100 nanograms per kg bodyweight
69. nd vector may result in the formation of a replication competent virus PER C6 X El gene from E1 deleted L1 9eorj Zero risk of replica adenovirus adenoviral vector tion competent virus with no sequence particle formation overlap PA317 Gag pol env from Retroviral vector L2 8eorj Small risk of RCR amphotrophic lacking the formation though mouse retrovirus gag pol env not unlikely genes Sequence overlap occurs in many cases psi CRE Gag pol and env Retroviral vector L1 7eorj Risk of RCR forma genes from lacking the tion is smaller than ecotropic mouse gag pol env with PA317 retrovirus as two genes Moreover the virus Separate constructs is ecotropic and therefore unable to infect human cells Phoenix E1 E4 from Retroviral vector L2 8eorj Risk of RCR forma ampho adenovirus SV40 lacking the tion is smaller than large T and Gag pol env with PA317 but not episomal gag pol genes zero due to the epi and env genes from amphotrophic mouse retrovirus as applied in the schemes of annex 2 somal presence of gag pol and env in multiple copies on the assumption that non hazardous inserts are used hazardous inserts would require a higher risk class ze xU BY Working with commonly used laboratory organisms 41 x lt iu 42 Laboratory requirements For generating adenoviral or mouse retroviral particles whether they are replication defec tive or not a L2 laboratory is recommended In addition a class I
70. ng GMOs Disturbance of an ecological balance may happen when a GMO pos sessing a certain characteristic is accidentally spread to the environment or when gene tic material originating from that organism spreads to other organisms in the environ ment The potential hazards of recombinant DNA technology and the risk assessment of activities involving this technology will be discussed more in detail in the following chapters of this booklet Other harmful effects Sometimes there are other unwanted effects that urge one to be even more cautious when handling biological material It is not possible to give an exhaustive list of these effects What matters is that one stops to think about the characteristics of biological material before starting to work with it One important class of genes that should be looked at carefully are genes that produce proteins with immune modulating properties although not all immune modulations are harmful For certainty about the possible level of harm the effects of the immune modulation should be thought through care fully and quite often consultation with experts will be necessary One example is the handling of a vacciniavirus in which a gene responsible for immune suppression is clo ned Immune suppression may lead to the body not being able to fight an infection by the virus In some exceptional cases infections with vacciniaviruses may lead to fatal encephalitis Biosafety x aad 7 AT 8 s
71. nment level li L2 L3 L4 26 Microbiological safety cabinet not required optional required required class II class 1 or Il class 1 or 11 in case of class Il an isolation suit should be used fitted with positive airpressure 27 Autoclave if autoclave in the building in the laboratory or In the laboratory then on site in an adjacent room 28 Double ended autoclave not required not required optional required 29 Centrifuge in the containment zone not required required required required not required if leakfree tubes are used 30 Vacuumgenerator fitted with a not required not required recommended required HEPA filter 3 Transfer of material to an autoclave situated outside the laboratory should occur according equivalent protection level Biosafety in the laboratory to a validated method that guarantees an 3 Working practices and waste management Specifications Containment level Li im L3 L4 31 Restricted access recommended required required and required and control control 32 Notification on the door recommended required required required a Biohazard symbol b a b c a b c d e f a b c d e f b co ordinates of the person in charge c containment level d type of biolocal risk e list of persons who have access f criteria for admittance 33 Labor
72. ns on the biosafety required required required required procedures 50 Efficient vector control not required recommended required required e g for rodents and insects 51 Circulation of animals forbidden forbidden forbidden forbidden 52 In case of manipulation of zoopathogens not required not required recommended period required period period during which contact of the to be specified in to be specified in personnel with the host animals should the authorisation the authorisation be avoided WASTE MANAGEMENT 53 Inactivation of biological waste and or required required required required biological residues using an appropriate validated method before dumping 54 Inactivation of contaminated material required required required required glassware etc using an appropriate and validated method before reuse or destroying 55 Inactivation of the effluents of sinks and not required not required optional required showers using an appropriate and validated method before dumping 4 5 Suitable footwear optional Full change of clothing and shoes before entrance and leaving Containment requirements hee 49 Belgian requirements for A1 A4 animal houses 1 Equipment and technical requirements Specifications Containment level Al A2 A3 A4 1 The animalarium is separated from not required required
73. o te beginning work Allow the cabinet to run for 10 minutes before beginning work Decontaminate the work surface and air intake grilles with a disinfectant such as 70 ethanol Only place materials and equipment in the cabinet which are required for immediate work Never place objects over the front or rear intake grilles Remember to always work from a clean to a dirty side On the dirty side you should place a small container for contaminated items such as pipette tips Move your arms slowly in a manner that will minimise the disruption of the airflow Perform all work well inside the cabinet not half outside it Never use a Bunsen burner since this may create turbulence in the airflow Use disposable inoculation needles instead 10 When your work is finished decontaminate all materials on the outside 11 before removing them from the cabinet Decontaminate the working surface and the air intake grilles 12 Allow the cabinet to run for another 5 minutes before turning it off 13 Close the cabinet lid Containment a combination of infrastructure and working practices f x ART 23 Class III safety cabinets A class Ill safety cabinet provides maximum protection to the worker and the environ ment The experiment however is less protected because there is no downward air flow inside the cabinet A class Ill cabinet is completely sealed and arm length rubber gloves are attached to po
74. ogenic and or genetically modified organisms have been defined Containment is achieved through a combination of physical containment measures and safe working practices Containment of hazards physical containment safe working practices walls doors safety cabinets no eating drinking or smoking working surfaces etc washing your hands etc Each subsequent containment level i e from level 1 to 2 2 to 3 etc requires a number of additional containment measures and safe working practices These containment levels have not only been formulated for laboratories but also for greenhouses animal houses and large scale process installations The annexes to this booklet contain an overview of the containment measures required in laboratories animal houses and greenhouses The requirements for large scale process installations and hospital rooms can be requested from your biosafety coordinator or at www biosafety be ventilation AB LE dimi closed door desinfection of benches Figure 6 Containment in a laboratory Biosafety in the laboratory Containment at the source As is the case for any other activity that may present a risk one should always try to tackle the risk at its source Among other things this means that one should always opt for orga nisms host organisms or vectors presenting less risk but providing the same results as their alternatives that do pose a threat I
75. on If no proper containment measures are taken pollen may be disseminated to the environment through the air or aided by insects Whether or not this presents a genuine risk depends on how the plant reproduces by self pollination or by cross pollination The spread of pollen by strict self pollinators has no effect what soever but when a cross pollinator is involved it should be carefully checked whether any of its wild relatives which it might successfully hybridize with is growing in the vicinity In addition to pollen seeds originating from transgenic plants may sometimes easily be disseminated in the environment Especially when they are very small or sticky these seeds are very likely to be accidentally taken along by a researcher leaving the growth chamber or greenhouse It is not only pollen or seeds that may be responsible for the undesired spread of transge nic plants Some plant parts may grow and turn into whole new plants themselves These reproductive parts of plants should not be discarded without destroying them properly For example the branch of a willow can grow roots and leaves very easily and the stembase of a cabbage can also grow roots This is why laboratory staff handling transgenic plants or plant material should pay special attention to the possible spread of plant parts that are still able to reproduce If there is a genuine possibility that a transgenic plant will be able to establish itself in the environment or th
76. on procedures chapter 6 of this booklet A possible variation to this example Cloning the chymosin gene into a self transmissible plasmid in E coli JM109 and transferring the modified bacteria onto a plastic matrix during which procedure the formation of aerosols cannot be avoided In this case the content of step 2 changes the vector does not meet the risk class 1 requirements As a consequence the second category of the first direction of annex 2 chapter A now becomes applicable instead of the first one The gene product is still well known and non hazardous As a result category j of the second direction remains applicable and the corresponding containment level is L2 What is more a very specific aerosol producing procedure is being performed for which an additional containment measure is required on top of the standard L2 measures the aerosol producing manipulations should be performed in a class Il safety cabinet The eventual contain ment level is therefore L2 supplemented with the use of a class Il safety cabinet This booklet contains the following information on this topic 12 containment requirements annex 1 to this booklet MA Working with commonly used laboratory organisms 37 Description of how to use a class Il safety cabinet chapter 5 of this booklet Decontamination and inactivation procedures chapter 6 of this booklet Are there any cases requiring special attention with regard to ad
77. ormation of autonomously replicating virusparticles i The sequence contains genetic Resp 14 L3 L2 Resp 13 L2 L2 information that codes for a hazardous gene product other than in f j The sequence does not contain genetic Resp L4 L3 L2 Resp 13 L2 L1 information that codes for a hazardous gene product The risk classification may in some cases have to be higher than given above in the case of the formation of an infectious virusparticle with an increased host cell range or an increased virulence or pathogenicity s What constitutes a hazardous gene product is the most difficult risk classification question Virulence gene products might be an example of a uA hazardous gene product Final risk classification depends on the suspected effect of the gene product in the used hostorganism Ate 60 Biosafety in the laboratory 4 Activities using plant cells Second direction Activities in which genetic material is or has been added that has not been characterised Activities in which characterised genetic material is or has been added First direction f The sequence contains genetic information T3 that codes for the production of a toxin of T2 respectively class Ti g The sequence contains genetic information Risk group 4 for the formation of a for eucaryotic cells Risk group 3 infectious virus of respectively risk group 4 Risk group 2 3 or 2 and the viral sequences that
78. purpurea Fusarium arthrosporioides Fusarium coeruleum Fusarium culmorum Fusarium graminum Fusarium oxysporum f sp betae Fusarium oxysporum f sp lycopersici Fusarium oxysporum f sp pisi Fusarium oxysporum f sp trifolii Fusarium solani f sp cucurbitae Fusarium solani f sp phaseoli Fusarium solani f sp pisi Glomerella cingulata anamorph Colletotrichum gloeosporioides Glomerella graminicola anamorph Colletotrichum graminicola Fytopathogenic parasites P ei Heterodera glycines Glomerella tucamanensis anamorph Colletotrichum falcatum Mucor circinelloides Mucor piriformis Mucor racemosus Mucor strictus Penicillium corymbiferum Penicillium cyclopium Penicillium digitatum Penicillium expansum Penicillium italicum Phytophthora infestans Phytophthora megasperma Rhizoctonia carotae Rhizoctonia fragariae Rhizoctonia tuliparum Rhizopus arrhizus Rhizopus stolonifer Sclerophthora macrospora Sclerospora graminicola Sclerotinia minor Sclerotinia trifoliorum Septoria apiicola Septoria azaleae Septoria chrysanthemella Septoria lactucae Septoria lycopersici var lycopersici Note This is a shortened list of pathogens If your organism is not on this list first consult the lists Lose at www biosafety be before concluding that your organism is not pathogenic bee The risk groups of some relevant pathogens 67 eee hee 68 We hereby show our gratitude to the following persons that have made us
79. required required other working zones in the same building or is located in a separate building 2 Entry via an airlock not required recommended required required with three compartments Doors that can be locked required required required required 4 Doors that close automatically not required required required required Windows that cannot be opened not required not required required required buth windows should able to withstand be closed during breaking experimentation 6 Airtight laboratory that enables not required optional required required decontamination with a gas 7 Building designed in such a way recommended required required required that accidental escape of animals is avoided 8 Observation window or equivalent recommended recommended required required system that enables one to see who is working in the laboratory 9 Decontamination facilities for the required sinks required sinks required sinks near required sinks and personnel the exit or in the shower the latter fitted airlock shower with a chemical sprinkler recommended in case use is made of isolation suit fitted with positive air pressure 10 Sinks that can be operated not required recommended required required non manually 11 Coathooks or changing room for required required required required the protective clothing 12 Tubing fitted with a system that not required not required recommended required prevents backward flow of fluids 13 Separ
80. riate method to this end Note Never work with autologous cells Especially when genes encoding for growth factors or oncogenes would be cloned into these cells contamination could have detrimental effects The reason for this is that the immune system would not recognise the cells as foreign Biosafety in the laboratory Working with genetically modified viruses or viral vectors Classification Genetically modified viruses are generally classified in the same risk class as their biological counterparts see the annexes to Vlarem 11 www biosafety be unless the insert increases the risk In the latter case the modified virus will be classified at a higher risk level than its biological counterpart The use of viruses and viral vectors cannot be detached from the use of cell lines This implies that here too the combination of viral sequences in the vec tor and in the cell line should be considered To produce replication defective virus parti cles cell lines containing the missing viral sequences needed to create virus particles are used Only when there is no risk that replication competent virus particles might be for med the virus can be classified at a lower level Packaging Viral sequences cell line in the cell line Vector by way Classification Classification Comments of example om rule El and piece of E1 deleted 8eorj Combination of E4 gene from adenoviral viral sequences in adenovirus vector cell line a
81. rom a pathogen that is hazardous for humans or the environment and of which the risks are not yet known The plants that carry a fytopathogen or GMO of biological risk class 4 Containment level G4 The plants that carry a virus of biological risk class 4 that poses an enormous threat to the environ ment or for which there is a zero tolerance of that carry a gene that is responsible for the produc tion of a substance that is very hazardous for humans or animals The plants that carry a fytopathogen or a GMO of biological risk class 4 that poses an enormous threat to the environment or for which there is a zero tolerance The plants that carry transgenes originating from a for humans animals or the environment very dangerous pathogenic organism and of which the risks are not known Biosafety in the laboratory CHAPTER C GUIDELINES FOR THE CLASSIFICATION OF ACTIVITIES IN ANIMAL HOUSES INVOLVING TRANSGENIC ANIMALS OR ANIMALS THAT CARRY GENETICALLY MODIFIED MICRO ORGANISMS OR CELLS Containment level A1 The animals that cannot spread easily themselves pigs sheep cattle and that have taken up a trans gene in their genome without the help of a viral vector The animals that cannot spread easily themselves and that carry a risk class 1 GMO Containment level A2 The animals that are able to spread easily themselves small rodents rabbits insects fish and that have taken up a transgene in their genome wi
82. rts in the cabinet in a gas tight manner to allow for manipulation of the materials isolated inside Materials are brought into and removed from the cabinet through a small air lock or double ended autoclave A glove ports with O ring for attaching arm lenght gloves to cabinet sash exhaust HEPA filter supply HEPA filter double ended autoclave or pass through box a Y ae V VLA OO D Room air F m G x B contaminated air 3 HEPA filtered air Horizontal laminar air flow cabinets and cross flow cabinets In laboratories where animal cells or in vitro plant materials are manipulated use is some times made of a horizontal laminar air flow cabinet or a cross flow cabinet These are cabinets in which sterile air is blown into the cabinet whereas the contaminated air is blown into the laboratory space This means that the laboratory worker is sitting in the contaminated air flow In other words these cabinets provide protection neither to the worker nor to the environment They are therefore not considered as real safety cabinets and their use should be discouraged in most cases These cabinets cannot be used for activities posing a risk to the worker or to the environment The use of such cabinets may be permitted exceptionally when no use is made of open containers holding GMOs such as open petri dishes containing modified bacteria or opened Erlenmeyer flasks containing bacterial cultures Bio
83. s 23 HEPA filtration of the air not require not required required on inward and extracted air 24 System that allows changing of the filters without required contamination of the environment 10 12 n surface water to come in and with doors that close automatically and can be locked With structure the walls roof and floor are meant TI e greenhouse should be a permanent construction with a seamless water tight covering situated on a premises such that it is impossible for If transmission via soil is possible In case of use of virus not retained by HEPA filtration special appropriate measures must be taken with regard to the extract air Containment requirements he 53 2 Safety equipment Specifications Containment levels GI G2 G3 25 Autoclave on site in the building in the greenhouse 26 Double ended autoclave not required not required optional 27 Fumigationroom or immersion bath for not required not required optional the transfer of living materials 3 Working practices and waste management Specifications Containment levels GI G2 63 28 Restricted access required required required and control 29 Notification of the biological risk not required required required 30 Specific equipment not required not required required 31 Protective clothing req
84. safety in the laboratory L Room air B contaminated air 3 HEPA filtered air A front opening J B sash crossflowcabinet C supply HEPA filter horizontal D blower laminar air flow cabinet Safe working practices Physical containment alone is not enough to create safe working conditions It should be combined with safe working practices Specific working practices have been defined for each containment level Bas ki leas ie Miroi Tedi As far as level 1 containment is concerned the working practices contribute most to cre ating safe working conditions These basic working practices are called Safe Microbiological Techniques SMT Everyone that manipulates micro organisms and cells in a laboratory should make it a daily routine to follow these procedures Containment a combination of infrastructure and working practices 25 Safe Microbiological Techniques 1 10 Keep doors and windows closed when experiments are in progress Wear a laboratory coat Eating drinking smoking and storing food and drinks are not permitted in the laboratory Do not wear jewelry and or a watch keep your hands clean and your nails short at all times Decontaminate any spills of GMOs immediately e remove fluids with tissues or paper towels and throw these into the waste basket for biologically contaminated waste decontaminate the surface on which the material was spilled with 70 ethanol or another validated disinf
85. se guideli nes are not legally binding but following these guidelines and the risk assessment princi ples set out above will facilitate a good implementation of the legal requirements After determining a first classification and depending on the type and scale of activity it should also be determined whether or not specific extra measures will be necessary in addition to the basic requirements of that containment level or whether some measures can be left out The legal requirements for the four containment levels in laboratories ani mal houses and greenhouses are listed in annex 1 Below a comparison is given between the categorisation of pathogens and GMOs in their tisk classes and the accompanying basic containment levels Pathogens Basic containment level Note level 1 gives requirements for both the infrastructure and the working practices although the requirements for the infrastructure are very limited SMP Safe Microbiological Practices only describes requirements for the working practices Classification and risk assessment wel 13 ae 4 THE Aa or AU Natural routes of infection Pathogens all have their own route of infection by which they spread from one host organism to another The table below lists a number of important routes of infection Route of infection All these routes of infection may depending on the type of work that is being performed occur in the laboratory As regards or
86. st environmentally friendly method In practice the fluids containing chlorine are poured down the drain all too often Principly fluids containing substances that might affect the proper functioning of a water purification plant should not end up in the com pany s waste water For a decontaminant to be effective it is also very important that it is in contact with the micro organisms long enough i e for at least 15 to 30 minutes in general Biosafety in the laboratory The appropriate decontamination and inactivation method for some relevant organisms Ss Decontamination Inactivation 1 2 70 Formal Wet heat Dry heat DETUR glutar ethanol dehyd 121 C 20 aldehyd only applies to small volumes of serum only for heat labile antigen vaccinia Biological waste must be disposed of An important distinction should be made between biological waste that has been inactivated before disposal and biological waste that has not been inactivated before disposal The latter has to be treated as hazardous medical waste and should be transported to an incinerator that is suited for the incineration of hazardous medical waste Biological waste includes all genetically modified and or pathogenic biological material cell cultures cultures of micro organisms tissues blood etc e typical laboratory waste of organic origin gels etc e all kinds of biologically contaminated material gloves paper tissues disposable cu
87. t chemical substances with different modes of actions against micro organisms They can contain oxidising chemicals quaternary ammonium salts surfactants and other substances These disinfectants can be used for decontamination as well provided that they are validated for the types of micro organisms you work with Do not only check the user manual of these disinfectants but also check what substances in what concentration are present and what modes of actions are combined in the disinfectant Decontamination especially the decontamination of surfaces can never be 100 effecti ve Decontamination can only result in a sharp reduction of the number of viable micro organisms a well performed bacterial decontamination will lead to a 90 reduction viral decontamination is often more effective What is more after the decontamination the remains of micro organisms often stay present This is why it is important to use hot water and soap to clean the work surfaces floors and door knobs during the weekly cleaning of the laboratory You need to decontaminate twice in order to decontaminate successfully Contamination accidents decontamination and inactivation f ze x 31 The effectiveness of different decontaminants Fungi bacteria mycobacteria lipid non lipid viruses viruses above 40 C V variable Inactivation f nt Ae It is often quite difficult to destroy biological material Bacterial spores for ins
88. tainers before transport not required required required 47 Control of contaminated run off water optional minimise prevent run off run off WASTE MANAGEMENT 48 Inactivation of biological waste and or biological required required required residues using an appropriate and validated method S before dumping beg If transmission via soil is possible 54 Biosafety in the laboratory 41 Inactivation of contaminated material glassware etc using an appropriate and validated method before reuse or destroying required required required 42 activation of the effluents of sinks and showers using an appropriate and validated method before dumping not required not required optional Containment requirements GUIDELINES FOR THE CLASSIFICATION OF MO ACTIVIT Introduction These guidelines are meant to offer help in the determination of the appropriate containment measures for activities with genetically modified organisms In this annex a distinction is made between activities involving micro organisms and cells chapter A activities with plants chapter B and activities with ani mals chapter C The transformation of plants using Agrobacterium tumefaciens is classified as an acti vity with the micro organism The use of these quidelines These guidelines offer especially help for determining the necessary containment measures for activities with micro organisms and cells chapter A of th
89. tance are resistant to temperatures of 100 C Sterilisation heating water under pressure at a tem perature of 121 C for 20 minutes is the recommended method in such cases Sometimes the water is even heated at temperatures of 134 C This is a very effective way to inactiva te micro organisms provided that the steam can reach all areas in need of sterilisation Air pockets are a well known problem Even animal cells are known to be able to survive in an air pocket during the sterilisation process A sterilisation tank should be filled in a very careful manner and all caps should be loosened Sterilisation is the recommended method to kill bacteria yeasts and fungi For other organisms like plant and animal cells and some viruses simpler methods are available Heating up to 80 C or exposing the cells to strong detergents is often sufficient to kill them It is however highly important to check whether the method to be used is a validated one in other words whether the organisms will be effectively destroyed Inactivation is much more effective in killing micro organisms than superficial decontamination if well performed inactivation can be 100 effective When bacteria present in fluids need to be killed sodium hypochlorite solutions are often used It is important to make sure that the final level of chlorine in such solutions is high enough to kill the bacteria 20 100ml I It should be mentioned that sterilisation is still the mo
90. tem grooving virus Barley yellow mosaic virus Beet western yellows virus Carnation ringspot virus Cucumber mosaic virus Hop american latent virus Hop mosaic virus Lettuce mosaic virus Maize dwarf mosaic virus Melon necrotic spot virus Papaya ringspot virus Pea early browning virus Potato leafroll virus Potato virus A Fytopathogenic bacteria and related organisms P Potato virus M Potato virus S Potato virus X Potato virus Y Tobacco mosaic virus Tobacco necrosis virus Tobacco rattle virus Tobacco streak virus Tobacco stunt virus Tomato bushy stunt virus Tomato mosaic virus Tomato yellow leaf curf virus Tulip breaking virus Turnip crinkle virus Turnip mosaic virus Wheat dwarf virus Wheat spindle steak mosaic virus Agrobacterium rhizogenes Agrobacterium rubi Agrobacterium tumefaciens Erwinia carotovora subsp betavasculorum Erwinia chrysanthemi pv chrysanthemi Erwinia salicis Erwinia tracheiphila Pseudomonas cichorii Fytopathogenic fungi P Pseudomonas fluorescens Pseudomonas syringae pv phaseolicola Pseudomonas syringae pv pisi Pseudomonas syringae subsp syringae Rhodococcus fascians Xanthomonas campestris pv aberrans Xanthomonas campestris pv alfalfae Xanthomonas populi Alternaria dauci Alternaria solani Botrytis allii Botrytis elliptica Botrytis fabae Botrytis hyacynthi Botrytis tulipae Cladosporium phlei Cladosporium variabile Claviceps gigantea Claviceps
91. thout the help of a viral vector The animals that are able to spread themselves and that carry a risk class 1 or 2 GMO Notes The classification of animals that carry genetically modified micro organisms or cells is more complex than the classification of the genetically modified micro organisms or cells as such This is because with such activities not only the risk class of the modified micro organism is important but also the combi nation animal micro organism the way the micro organism is administered and the possibilities of the micro organism to spread from the animal to the environment In a normal laboratory the micro orga nism is kept in a closed container and if this container is opened and there is a risk of spread to the environment then in many cases the container will only be opened in a safety cabinet class 2 and hig her The laboratory animal cannot be considered a closed container This is why for every specific com bination of laboratory animal micro organism it has to be determined whether there is a need for addi tional measures to prevent escape of the micro organism to the environment In annex 1 under con tainment levels A1 to A4 these additional measures have been described under recommended or optional An example a risk class 2 genetically modified adenovirus should be handled in a laboratory using L2 containment measures When such a virus is vaporised as an aerosol into the lungs of a mouse basi
92. tory is sufficient for cells like NIH 3T3 In practice cells like these are usually inoculated and grown in well equipped cell culture facilities that meet more than the basic L1 requi rements A class Il safety cabinet is usually present to protect the cells against infectious agents when they are handled in the open Instead of class Il safety cabinets horizontal laminar flow cabinets are often used The latter are suited for level 1 activities with cells that do not involve infectious agents They are however not sufficient for manipulations with cells belonging to L2 In that case a class Il safety cabinet is required to protect both the laboratory worker the environment and the experiment In comparison with those of L1 the L2 requirements are more stringent with regard to access to the laboratory and activities that might lead to the creation of aerosols Waste All the biological waste originating from genetically modified cells needs to be inactivated However a distinction should be made between cells requiring L1 containment and those requiring L2 containment As far as L1 cell cultures are concerned it is sufficient to expo se them to a strong detergent for a period of time and then pour it out in the sink Contaminated materials can be dealt with in the same manner L2 cell cultures require more rigorous measures to ensure that any hazardous biological agents present in the material are killed as well Sterilisation is the most approp
93. uire recommended required 8 Structure waterimpermeable and easy to clean not require recommended required 9 Windows that cannot be opened not require not required required 10 Airtight room that permits decontamination with not required not required required a gas 11 Decontamination facilities for the personnel required required required sinks in the sinks sinks airlock or near the exit shower optional 12 Sinks that can be operated non manually not require not required required 13 Tubing fitted with a system that prevents backward not required not required recommended flow of fluids 14 Surfaces resistant to acids alkalines organic solvents not require recommended required and desinfectants 15 Waterimpermeable floor not requirei recommended required 16 Management of flow of contaminated water optional minimise flow prevent flow 17 Autonomous electricity backup system not required not required required 18 Fire alarm system not taking into account local not require optional required fire regulations 19 System to communicate to the outside world not require optional required VENTILATION 20 Input and extract airflowsystem interconnected to not required optional required prevent accidental overpressure 21 Input and extract airflowsystem can be closed not required optional required using valves 22 Negative airpressure in the controlled zone when not require not required optional compard to adjacent zone
94. uired required required and specific for the containment zone 32 Decontamination of the protective clothing not required not required required before it leaves the containment zone 33 Gloves not required optional optional 34 Shoe coverings or decontamination bath for not required optional optional the shoes 35 Generation of aerosols minimise minimise prevent 36 Specific measures to prevent the spread of aerosols not required recommended required 37 Mechanical pipetting required required require 38 No drinking eating or smoking no use of cosmetics required required require no manipulation of contacts or the stocking of consumables 39 Register in which all activities are notified required required require transport of plant material inoculation of GMM s etc 40 Verification of control measures and protective required required require equipment 41 Notice for the use of decontaminants required required require 42 Instruction of the personnel required required require 43 Written instructions on the biosafety procedures required required required 44 Circulation of animals forbidden forbidden forbidden 45 Measures to control undesired species such as insects required required require rodents arthropods 46 Selfspreading organisms transport in the installation between container container double containment zones optional container note in the register not required recommended required decontamination of con
95. us epidermidis Streptobacillus moniliformis Streptococcus agalactiae Streptococcus dysgalactiae Klebsiella oxytoca Streptococcus equi Klebsiella pneumoniae Streptococcus pneumoniae Klebsiella spp Streptococcus pyogenes Streptococcus spp Streptococcus suis Streptococcus uberis Vibrio cholerae including El Tor Yersinia pestis Listeria ivanovii Listeria monocytogenes Mycobacterium avium subsp paratuberculosis Mycobacterium bovis except the BCG strain a cx Note This is a shortened list of pathogens If your organism is not on this list first consult the lists DO at www biosafety be before concluding that your organism is not pathogenic 64 Biosafety in the laboratory Human and animal pathogenic viruses H A Adenoviridae Animal adenoviruses Human adenoviruses African swine fever virus Arenaviridae Flexal virus Junin virus Lassa virus Machupo virus Equine arteritis Simian haemorrhagic fever virus Astroviridae Astroviruses Baculoviridae Invertebrate baculoviruses Birnaviridae Drosophila X virus Infectious pancreatic necrosis virus Bunyaviridae California encephalitis virus Hantaan virus Korean haemorrhagic fever Filoviridae Ebola virus Marburg virus Flaviviridae Dengue virus 1 4 Japanese encephalitis virus Yellow fever virus Hepatitis C virus Border disease virus Bovine diarrhoea virus Hog cholera virus Herpesviridae Avian herpesvirus 1 ILT Marek s disease Bov
96. ve been classified as pathogens in the risk groups 4 3 and 2 as described in the annexes of Vlarem Il Guidelines for the classification of GMO activities Lose de 57 1 Activities with host vectorsystems that do or do not fulfill the criteria for categorisation in risk class 1 and activities with non viral pathogenic hostorganisms First direction pathogens The host is a pathogen of respecti fulfill the criteria for categorisation in risk vely risk group 4 3 or 2 class 1 a Second direction 2 Activities in a host vectorsystem that does not 3 Activities in pathogenic hosts excluding viral Activities in which genetic material is or has been added that has not been characterised he sequence contains genetic information that codes for a toxin of respectively class g The sequence contains genetic information for the formation of a for eucaryotic cells infectious virus of respectively h The sequence contains genetic information for the formation of a defective for eucaryotic cells infectous virus of respectively i The sequence contains genetic information that codes for a hazardous Risk group 3 Risk group 2 Risk group 4 gene product other than in f j The sequence does not contain genetic information that codes for a hazardous gene product Activities in which characterised genetic material is or has been added
97. ww biosafety be H risk group for humans A risk group for animals P risk group for plants OP opportunistic pathogen have a limited risk of infection because they are not able to spread via air virus of which the biological risk depends on the hostanimal Human and animal pathogens bacteria and related organisms H A H A Mycobacterium leprae Mycobacterium tuberculosis Neisseira gonorrhoeae Neisseira meningitidis Neisseira spp Pseudomonas aeruginosa Salmonella Abortusequi Salmonella Abortusovis Salmonella choleraesuis enterica subsp arizonae Salmonella Dublin Salmonella Enteritidis Salmonella Gallinarum Bacillus anthracis Bacillus cereus Bacillus lentimorbus Bacillus popiliae Bacillus sphaericus Bacillus thuringiensis Bordetella avium Bordetella bronchiseptica Bordetella parapertussis Bordetella pertussis Campylobacter coli Campylobacter fetus subsp fetus Campylobacter fetus subsp venerealis Campylobacter jejuni Salmonella Paratyphi A B C Campylobacter spp Salmonella Pullorum Chlamydia pneumoniae Salmonella Typhi Clostridium botulinum Salmonella Typhimurium Clostridium tetani Shigella boydii Enterobacter spp Shigella dysenteriae Type 1 Enterococcus faecalis Shigella flexneri Escherichia coli excluding non pathogenic strains Helicobacter hepaticus Helicobacter pylori Klebsiella mobilis Enterobacter aerogenes Shigella sonnei Staphylococcus aureus Staphylococc

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