Bio  GM Safety Working to the Code

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Regulations. COSHH GM Contained Use RegsSpecific approval required Anti Terrorism Crime

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Bio GM Safety Working to the Code

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1. Bio & GM Safety Working to the Code Ann Hallam University Biological Safety Adviser

2. Regulations COSHH GM Contained Use Regs Specific approval required Anti Terrorism Crime & Security Act Schedule 5 list – pathogens & toxins

3. Biological Agents COSHH Definition Any of the the following if they can cause infection, allergy, toxicity or other human harm. micro-organism cell culture human endoparasite

4. Classification ACDP 4th Edition 1995 “Categorisation of Biological Agents According to Hazard and Categories of Containment.” HSE Approved List - schedule to COSHH Bacteria, Viruses, Parasites & Fungi Hazard grouping 1 - 4 (low to high) Containment facilities/lab standards (1 - 4) List identifies control measures to be applied

5. Control measures = containment level Increasing levels of stringency CL 1 – 4 Prescribes lab facilities Access controls Use of Microbiological Safety cabinets Provision of S O Ps Disinfection & Decontamination regimes Information instruction & training PPE requirements

6. Biological Agents Hazard Group 1 Unlikely to cause human disease Animal tissues and cell lines Well established human cell lines - history of safe use e.g. MRC 5 Plant cells/materials Assign to CL 1

7. Hazard Group 2 Can cause disease May be a hazard to employees Unlikely to spread to community Prevention or treatment available Bacillus cereus, Clostridium spp, campylobacter Most wild type E. coli, Pseudomonas aeruginosa, Proteus vulgaris, Staph aureus. Human tissues & primary cell cultures Assign to CL 2

8. Hazard Group 3 Can cause severe human disease Serious risk to employees May spread to community Prevention or treatment available Anthrax; Brucella abortus/canis/suis; E.coli O157 Mycobacterium bovis/leprae/tuberculosis; Salmonella typhi/paratyphi; Yersinia pestis. HIV; SIV; Hepatitis; Hantaan Plasmodium faliciparum, Trypanosoma cruzi Assign to CL 3

9. Hazard Group 4 Severe human disease – likely to spread – no treatment Virusus such as Lassa fever Rabies Congo heamorrahagic fever Ebola Marburg Variola CANNOT BE USED HERE

11. Containment level 2 - facilities. Bench - impervious, washable, chemical resistant. Floor - coved, continuous, sealed. Wash-hand basin by the door. Negative pressure to corridor - [mechanical ventilation]. Restricted access, door kept closed. Autoclave - in building Lab coat storage - enough for occupants

20. Centrifuges Use sealed buckets or rotors Balancing Check seals before use CL2 & 3 - open in safety cabinet Clean and disinfect centrifuge and rotor after use. Some disinfectants attack metal rotors! Spillage/breakage procedure

21. Microbiological Safety Cabinets Class I to III Classes DO NOT relate to containment level!! Class III - highest protection Class I - good general operator protection Class II - combines protection of work and worker against contamination.

22. Video Training Safe use of Microbiological safety cabinets Access at http://webct6.is.nottingham.ac.uk/webct/entryPage.dowebct Log in, view video & supplementary info Complete on-line assessment

23. Laminar Flow Hoods DO NOT confuse laminar flow hoods with Microbiological Safety Cabinets. LFHs draw clean filtered air vertically or horizontally across the work to protect the work from external contamination. There is no worker protection as there is no inward air flow - horizontal units direct air towards the operator!!! Use only for non hazardous organisms

24. Unscreened human tissue and fluids Risk of “hidden” pathogens. CL2 if unknown, CL3 if known HG3 present. Very low aerosol risk - cuts, scratches, injection Hep B vacc. before start - contact Occ Health. Use screened/low risk group donors where possible Contamination of skin, mucous membranes, work area. Designate area, written protocols followed, Avoid sharps and glassware, Cover cuts with waterproof plasters, wear gloves, Rigorous decontamination procedures, MSC if aerosols produced - mixing, shaking, sonication Sharps Injury procedure & Ability trace source

25. SHARPS INJURY & EYE /MOUTH SPLASH PROCEDURE First Aid Encourage bleeding, do not suck Wash with soap & water Wash eye/mouth with water Retain sample if possible Report Incident Line manager/senior staff Complete accident report Contact OHD / duty microbiologist via QMC/CH switch

26. Hazard dependant on nature of cells. Non human/primate origin/well characterised/low risk of EI - minimal risk = CL1 Human primate cells not fully authenticated, primary cells of human/simian origin = CL 2 [ or above] Viral infected lines - CL appropriate to agent Be aware of spontaneous transformation - primary cultures Transformation signs = phenotype changes, [shape /size/cofluence/loss of CI/ speed of cell division Precautions Don’t culture or transform own/close colleagues cells Limit culture time - 48 - 72 hr max Use tissues from screened sources where possible Work with cell cultures

27. Video mammalian cell culture

28. Disinfectants - selection Type Spectrum of activity Specific activity for different micro-organisms Circumstances Dirty or clean - organic load intra or extracellular viruses Chemical incompatability Temperature, pH, hardness of water.

29. Disinfectants - selection Consider material/surfaces to be disinfected Metal equipment surfaces – avoid acids, alkalis, hypochlorites Plastics could be damaged by phenolics Spills – consider powder form, or gel absorbents Consider Hazardous properties toxic/corrosive [phenolics /hypochlorite] irritant [ Virkon , formaldehyde] Sensitising [ gutaraldehyde ] Reaction products [formaldehyde + Chlorine ]

30. Virkon [peroxygen compound] Broad spectrum of effectiveness Activity reduced by protein/salts WC = 1% discard jars/bug cultures 2% buffered systems Colour indication Stable for 7 days Does not bleach clothing Prolonged exposure can cause corrosion [10m] 1% working solutions are relatively safe Powder irritant

31. Trigene Halogenated tertiary amine+ surfactants Small efficacy spectrum WC = 2% discard jars, 10% body fluids/spills non toxic, less irritant than Virkon

32. Clorine - based hypochlorite chloros;presept Rapid action - Protein denaturation WC General use 1,000-2,500 ppm Discard containers 5,000-10,000 ppm Spills 20,000 ppm Chlorine produced if mixed with acid Carcinogens if mixed with formaldehyde Corrosive, damages metal . Limited shelf life - chemical reaction

33. Alcohols 70% ethanol; 60% iso- propanol Relatively poor efficacy. Susceptible to interference. Use on physically clean surfaces. Flammability risk - do not use sprays in MSCs

34. Disinfectants - USE Validation. HG 1 &2 - rely on manufacturer data - but use recommended concentrations. With HG3 organisms, or working off-spec with HG2 organisms, validate the effectiveness of the disinfection protocol under specific conditions of use. Formalise into lab disinfection procedures. Stability of working solutions. Effectiveness decays with time. Some (eg virkon) have colour indicator. Label with expiry date. Contact time. Check concentration and contact time necessary for your conditions - suppliers data.

35. Autoclaves Mandatory for HG 3 waste & certain HG 2/GM waste Portable/benchtop models not suitable for waste inactivation Transport - robust leakproof containers ID source of waste Written operating procedure Training and authorisation needed Visual check of seals and steam leaks pre-use Protective clothing - lab coat; impervious apron; heavy duty gauntlets; face visor, robust shoes NO RADIOACTIVE MATERIALS/ toxic chemicals Maintenance, validation & calibration

36. Autoclaves

37. Biohazard (Clinical) Waste Requirements Segregation Identification Packaging Yellow bags Label source - dept name tape 3/4 full max. Seal - tie, knot, proprietary clip. Remove to secure collection point Final Disposal - Incineration Infective wastes - autoclave first

38. Transport of Biological Material Regulations for road, rail and air. SEE University Code of Practice Classification, labelling and packaging by competent person. Infectious Substances Cat A & Cat B Package - UN approved to prevent release for Cat A Special package if dry-ice used. Shipper’s declaration for air transport. Advise use of Courier School/departmental safety officer must be consulted first.

39. Transport of Biological Material

41. GM - What is it? alteration of genetic material of an organism - PICR VIRUS RETROVIRUSES ADENOVIRUS VACCINIA LENTIVIRUS BACCULO VIRUS PICR VIRUS RETROVIRUSES ADENOVIRUS VACCINIA LENTIVIRUS BACCULO VIRUS

42. Some Examples Bacterial cloning using plasmid vector containing ‘foreign’ DNA insert Use of viral vectors containing DNA for use in mammalian expression systems Production of GM animals or plants [transgenics, knock outs, chimeras]

43. Any of these activities where you use any of these barriers to limit contact and provide protection to humans & environement Any of these activities where you use any of these barriers to limit contact and provide protection to humans & environement

44. Is it safe? In more than 25 years since start, no serious harm reported from accidental exposure to GMOs

45. Edinburgh Uni - Fined £3500 in 1999 for failure to keep adequate records and carry out suitable and sufficent risk assessments. Gary Burns [now at AZ] was the SI - prosecuting for HSE - Though low risk the technology moves quickly and situations can change rapidly - RA id very important. Birmingham Uni - Dept Cancer Studies - Early 90s - Prohibition Notice as level of containment used was not proportional to the degree of risk Imperial College London - July 1991 - fined £25000 + £21000 costs for failure to adequately protect staff and environment from release of hybrid virus - Hep C & Dengue fever. There was no vaccine available and the college failed to apply the procedures of good microbiological practice and good occupational safety and hygiene - Reg 17 of CU Regs. Edinburgh Uni - Fined £3500 in 1999 for failure to keep adequate records and carry out suitable and sufficent risk assessments. Gary Burns [now at AZ] was the SI - prosecuting for HSE - Though low risk the technology moves quickly and situations can change rapidly - RA id very important. Birmingham Uni - Dept Cancer Studies - Early 90s - Prohibition Notice as level of containment used was not proportional to the degree of risk Imperial College London - July 1991 - fined £25000 + £21000 costs for failure to adequately protect staff and environment from release of hybrid virus - Hep C & Dengue fever. There was no vaccine available and the college failed to apply the procedures of good microbiological practice and good occupational safety and hygiene - Reg 17 of CU Regs.

46. Main provisions of Regs Set up a GM Safety Committee to approve RAs Risk Assessment – humans and environment RA to be approved & recorded Assign containment level to protect H & E CL determines activity class. Prescribes standards for containment facilities Notify HSE [class 2 or above – Fee!!] Emergency plan Report certain incedents /accidents

47. What’s suitable and sufficient Depends on the complexity of the activity: Review Activity change - you increase the scale, change where the work is done When you find out new information about the GMO you have made - often this can result in reduction of CL - once it is characterised We ask for a minimum annual review - initiated by GMSC. Notification implication for presiously notified activities.- moving work to other GM registerted facitliy or where there is significant change to risk. Fee chargedWhat’s suitable and sufficient Depends on the complexity of the activity: Review Activity change - you increase the scale, change where the work is done When you find out new information about the GMO you have made - often this can result in reduction of CL - once it is characterised We ask for a minimum annual review - initiated by GMSC. Notification implication for presiously notified activities.- moving work to other GM registerted facitliy or where there is significant change to risk. Fee charged

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