GENERAL BIOSAFETY. Understanding the Regulatory Basis of the NIH Guidelines Working in a Biological Laboratory

Transcription

1 GENERAL BIOSAFETY Understanding the Regulatory Basis of the NIH Guidelines Working in a Biological Laboratory

2 General Biosafety Training Overview Introduction to the Biosafety Program (BSP) NIH and MIT policies regarding work with recombinant DNA (rdna), synthetic nucleic acids and biological materials Aspects of risk to consider when working with biological materials Biosafety levels 1 and 2 and corresponding work practices and engineering controls Emergency response for an incident involving biological materials, rdna or synthetic nucleic acids

3 Biosafety Program Services Risk assessment and biosafety support for research activities Management of campus-wide regulatory compliance Regulation Level of Oversight Description NIH Guidelines Federal Any institution receiving NIH funds is subject to these guidelines Cambridge General Ordinances (Chapter 8.20) Committee on the Assessment of Biohazards/Embryonic Stem Cell Research Oversight (CAB/ESCRO) City Institutional Requires all biotechnology research to abide by the NIH guidelines without exemption Committee formation required by NIH Guidelines for the purpose of local oversight of MIT research activities

4 The NIH Guidelines A Brief History Stemmed from concerns about safety/ethics of rdna research in the early 1970s 1973 Gordon Conference Voluntary moratorium on rdna research International conference held in Asilomar, CA rdna research deemed too important to restrict altogether Research to continue under well-defined guidelines Oversight at local level and in collaboration with the NIH Resulted in the first draft of the NIH Guidelines Guidelines were formally published in 1976 by the NIH s Office of Biotechnology Activities (OBA)

5 The NIH Guidelines Important Sections Section III Experiments covered by the Guidelines Section IV Responsibilities Biosafety Officer (BSO) IBC (CAB) Institution Investigator Appendix B Pathogen Risk Groups Appendix G Biosafety Levels

6 Biosafety Program Services Biological Research Registrations (BRR) protocol registration and review Support to the CAB/ESCRO Committee composed of MIT faculty, researchers, occupational health professionals, and community members appointed by the MIT president Approving body for research involving: Recombinant DNA and Synthetic Nucleic Acids Biological Agents Infectious agents Human materials Toxins of biological origins Animal research biosafety Human embryonic stem cells (hes) Induced pluripotent stem cells (ips) nanoparticle-based delivery systems for nucleic acids and drugs Laboratory inspection

7 Biosafety Program Services Support of other Institutional Committees Committee on Animal Care (CAC) Committee on the Use of Humans as Experimental Subjects (COUHES)

8 Biosafety Program Services Additional Regulations and Guidelines Regulation/Guidance Source Bloodborne Pathogen Standard Sanitary Code Chapter VIII Biosafety in Microbiological and Biomedical Laboratories (BMBL) Multiple regulations covering human embryonic stem cells (hesc) Select Agent Program OSHA MA CDC, NIH Federal and MA Description Dictates requirements for safe handling of human-derived materials Defines how medical and biological waste must be stored, treated, transported and disposed of Comprehensive laboratory biosafety guidance including risk assessment and agent information Guidelines for use and development of hesc CDC/APHIS BSP ensures exclusion from this program by controlling the amount of certain biological toxins used on campus

9 Biosafety Program Services Bloodborne Pathogen Program (OSHA) Indoor air quality Advice on sterilization Advice on biological waste management and disposal Shipping biological materials and rdna Import/export permits Biosafety cabinet consultation and training

10 What are some of the risks when working with biological agents and rdna? Infections of researchers or others Infections of domestic, wild, or agricultural animals or plants Invalidation of the experiment / contamination of lab stocks

11 What factors affect the risk of working with biological agents and rdna? Infectivity infectious dose vs. amount you are using Pathogenicity - health impact Availability of prophylaxis Before exposure (e.g. vaccination) After exposure but before infection (e.g. antibiotics, antivirals) Your health status Consult with MIT Occupational Heath or your personal physician about your research activities

12 Routes of Exposure and Transmissibility Injection / non-intact skin Mucous membranes (eyes, nose, mouth) Inhalation Ingestion These and other factors contribute to choice of biosafety level for research

13 What is a Biosafety Level? Based on a risk assessment A combination of lab practice / technique safety equipment facility design Based on concept of containment For protection of personnel lab environment environment outside lab

14 Certain experiments require BL1 physical containment and must follow prescribed laboratory practices.

15 Biosafety Level 1 Suitable for work involving well-characterized agents not known to cause disease in healthy adult humans and of minimal potential hazard to laboratory personnel and the environment. Bacillus subtilis, attenuated E. coli K-12, Saccharomyces cerevisiae Rodent cells or tissues (e.g. CHO, RAW264.7) Viral Vectors: Adeno-associated virus (AAV), baculovirus

16 Standard Microbiological Practices Restrict or limit access when working Use mechanical pipetting devices Open containers away from face Avoid contaminating the outside of containers Disinfect the outside of containers before work and before returning it to storage Wash hands!

17 Standard Microbiological Practices Minimize creation of splashes and aerosols Aerosol Droplet Size < 5 μm > 5 μm Visibility Invisible Often visible Travel distance > 3 ft < 3ft Behavior Similar to a gas, will not settle, removed by the HVAC system Gravity will cause droplets to settle on surfaces

18 Standard Microbiological Practices Minimize creation of splashes and aerosols Aerosols are easily inhaled Both contribute to surface contamination within the laboratory Evidence of droplets

19 Which lab activities create aerosols? Pipetting Centrifuging Cell lysis Blending / Mixing / vortexing Shaking Spills Sonicating Opening containers Inoculating animals intranasally Harvesting infected tissues from animals

20 Decontamination Decontaminate work surfaces after completing work and after any spill of biological material Choose the appropriate disinfectant for the biological material you are working with 10% Clorox Germicidal Bleach must be made weekly broadly effective against microorganisms including spores and non-enveloped viruses 70% Ethanol effective against most vegetative bacteria or enveloped viruses Biological toxins and prions require special decontamination procedures Consult BSP or go to the following link for more information

21 Prohibited Activities Eating (chewing gum) Drinking Smoking Applying cosmetics, i.e. chap stick, lotion Handling contact lenses Avoid touching face nail biting Mouth pipetting Storing food or drink

22 Personal Protective Equipment (PPE) Lab coat Gloves Your street clothes are part of your PPE!! Wear appropriate clothing while working in a laboratory.

23 PPE is to remain in the research area Carry your samples in a secondary container OR Use the one glove technique Parafilm Sealed Secondary Container Primary Container

24 Personal Protective Equipment (PPE) Additional PPE may be needed based on procedure or DLC requirements Face protection Eye protection Alternatives?

25 Hazard Communication: Signs and Labels Biosafety Level ( BL ) signs at lab entrances BL practices signs within labs Biohazard stickers on potentially contaminated items such as: Refrigerators/Freezers Incubators Centrifuges Storage Containers Waste Containers

26 Biohazardous Waste Researcher has responsibility for management of research material from cradle to grave Type of biological waste: 1. Liquid 2. Solid Regulated medical waste (i.e biowaste box) Solids (autoclave please see BSP for training and program information) 3. Sharps

27 Biohazardous Waste Disposal Liquid waste Liquid Waste Use chemical disinfectant Clorox bleach (1:10 final volume) Let sit for at least twenty minutes Pour down the drain and flush with water

28 Vacuum Line Protection A = primary collection flask with disinfectant B = overflow flask with disinfectant C = hydrophobic or HEPA filter D = to vacuum pump Bin = secondary containment for flasks

29 Biohazardous Waste Disposal Biowaste box program Biowaste box with plastic lid and base with casters, double red bag liners All solid biowaste including rdna is disposed in these containers These containers are collected by EHS from online requests and disposed via Stericyle. ent/biological-waste-pick

30 Bench-top Biowaste Transfer Container For Collection of: Conical tubes Microfuge tubes Serological pipets Pipet tips ALL GLOVES

31 Containers must be sealed and placed in the biowaste box when full. Needles & syringes Razor blades and scalpels Toothpicks Pasteur pipettes Glass vials, slides Broken glass Wire Anything else that can possibly puncture or cut the skin (contaminated or not) Please report problems or direct questions to EHS at No chemicals, reagents, solutions, or volatile substances No radioactivity No liquids No gloves No paper towels, kimwipes, wrappers, paper, cardboard, etc No plastic bottles, plastic tubing, plates, wells and other non-sharp plastic containers* * Note: Round bottom falcon tubes, petri dishes, microtiter plates and other non-sharp plastics should be collected in biowaste boxes.

32 Absolutely NO Sharps in the Regular Trash!

33 Experiments at the BL2 physical containment level require prescribed laboratory practices and containment equipment.

34 Biosafety Level 2 Suitable for work involving agents that pose moderate hazards to personnel and the environment. Organisms associated with disease that is rarely serious and often treatable Work with infectious/pathogenic agents i.e. Hepatitis B virus, Salmonella typhimurium, Staphylococcus aureus, Streptococcal spp. Work with human derived material including established and primary human cell lines/culture. Work with non-human primate tissue or cell cultures Viral Vectors: lentiviral vector, Adenoviral vector

35 BL2 Work Practices Standard Microbiological practices Access is restricted Reducing or eliminating sharps if possible

36 Engineering Controls Biosafety cabinet Disposable loops Safety cups for centrifuges Chemical fume hoods

37 Biosafety Cabinet: Class II, Type A2 Protects Lab personnel Lab environment Product Suitable for use with Biohazardous materials Cell culture Do NOT use Volatile toxic chemicals Flammable chemicals Radionuclides Open flame

38 Biosafety Cabinet: Procedures Purge cabinet before and after work Wipe down cabinet with appropriate disinfectant before and after use Check certification date and differential pressure gauge before use If these or a low-flow alarm indicate the cabinet is not working properly, discontinue use Do NOT block grille or airflow slots Minimize hand arm motion and take other measures to reduce disruption of airflow Keep glove-sleeve gap closed UV light is NOT adequate for disinfection Always use a chemical disinfectant

39 Biosafety Cabinet: Procedures Layout of Equipment: clean to dirty

40 Emergencies Sign up for MIT Alert* Program your cell phone (fire/ injury/ police 24/7) Dial 100 from any lab phone Use emergency response guide posted in each lab For EHS Office, call FIXIT (3-4948) after hours For EHS Office from 8 to 5, call ext * T h i s i s p a r t o f t h e I n s t i t u t e ' s c o m p r e h e n s i v e e m e r g e n c y n o t i f i c a t i o n s ys t e m. Y o u w i l l r e c e i v e a n S M S m e s s a g e o n yo u r c e l l p h o n e i n t h e e v e n t o f l i f e s a f e t y o r p u b l i c h e a l t h e m e r g e n c i e s.

41 What to do if you are exposed to a biological material or rdna If Direct contact to your face or skin or If stuck by a needle or sharp Immediately, wash the area with soap and water for minutes If it s in your eyes, nose or mouth, flush with water for 15 minutes Tell your supervisor immediately Go to the Medical Department (E23) immediately Supervisor must submit an incident report

42 MIT Medical Building E23 on Carlton St. Appointment hours: M-F 8:30a-5p Urgent Care: Daily 7a-11p Outside hours: Call

43 How to clean up a biohazardous spill (including biological material or rdna) 1. Assess the situation, alert others in the area, put on appropriate PPE, gather spill kit and appropriate equipment 2. Remove any broken glass or sharps with tongs or dust pan/broom into sharps container Large sharp material goes into a rigid container, e.g. cardboard box, sealed then into bio box 3. Cover the spill with paper towels 4. Disinfect the spill: Saturate paper towels with disinfectant, let sit for 20 minutes 5. Clean up the spill and dispose of spill materials in bio box 6. Repeat steps 2-5

44 What is wrong with this picture?

45 The End QUESTIONS?? Biosafety Program and researcher responsibilities Risk assessment for working with biological materials and recombinant or synthetic nucleic acids Biosafety levels work practices and engineering controls Emergency response

46 References Biosafety in Microbiological and Biomedical Laboratories, 5th edition NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules 105 CMR 480 MA State regulation regarding infectious waste Cambridge Public Health regulations on laboratory biosafety MIT Committee on the Assessment of Biohazards & Embryonic Stem Cell Research Oversight OSHA Bloodborne Pathogen Standard s/bmbl5/index.htm gs/105cmr480.pdf ervices/regulatoryactivities/biosafety/index.php disp.show_document?p_id=10051&p_t able=standards