Biological and Chemical Safety Manual

Fall, 2012

1 EMERGENCY INFORMATION (Fill out laboratory specific information and post outside of laboratory – copy to EHS dept.)

Principal Investigator

After -Hours Contact Information

Lab Location

Location Of Chemical Spill Kit

Location Of Biological Spill Kit

Location Of Fire Extinguisher

Location Of Fire Alarm

Location of Eye Wash Station

Location of Safety Shower

USD Environmental Health and Safety Chemical & Radiation Safety Kevin O’Kelley, Director of Environmental Health & Safety [email protected] , 605-677-6265

Institutional Review Board (IRB) Sandra Ellenbolt, Director of Human Subjects Protection [email protected] , 605-677-6067

Institutional Animal Care and Use Committee (IACUC) Peter Autenried, Director of Animal Resource Center [email protected] , 605-677-5174

Institutional Biosafety Committee (IBC) Victor Huber, Ph.D. [email protected] , 605-677-5163

Facilities Management Call Desk: 605-677-5341 (After hours, call USD Public Safety)

USD Public Safety Office: 605-677-5342

Fire/Police/Emergency Medical Services Dispatch: 9-911

2 Preface

A safe working and learning environment is an expectation of USD and is provided to all employees, students, and guests at the highest level reasonably possible. The University of South Dakota Biological and Chemical Safety Policy is designed as a reference for individual laboratories to provide a safe and productive work environment while complying with applicable federal and state rules and best practices. Each laboratory group should supplement this policy when necessary to insure health and safety of workers is not compromised.

The policy is divided into Chemical Safety and Biological Safety sections. Radiation Safety is addressed in the University of South Dakota’s Radiation Safety Policy for Authorized Workers.

The information in the Chemical Safety section is in accordance with guidance found in the National Research Council’s Prudent Practices in the Laboratory (National Academy Press, 1995) and the Occupational Safety and Health Administration (OSHA) Occupational Exposures to Hazardous Chemicals in Laboratories (29 CFR 1910.1450). The Chemical Safety section outlines the University of South Dakota’s Chemical Hygiene Plan.

The Biological Safety section, in conjunction with the Centers for Disease Control and Prevention (CDC) / National Institutes of Health (NIH) Biosafety in Microbiological and Biomedical Laboratories (BMBL), provides the requirements for safely working with biohazardous materials at The USD.

The primary concern when working with hazardous chemicals and pathogens is the safety of personnel working with these agents and the prevention of releases of these agents. While this guide is a sufficient starting point, some laboratories may need to supplement this guide with their own workplace specific standard operating procedures (SOPs).

The Principle Investigator (PI) has primary responsibility for ensuring the safety of students, faculty, staff, visitors and the environment with respect to their laboratory operations. The Environmental Health and Safety Office can provide guidance or information for questions regarding chemical and biological Safety.

3 Contents Scope and Applicability 5 General Laboratory Practices and Safety Equipment 7 Personal Protective Equipment (PPE) 10 Fume Hoods and Biosafety Cabinets 12 Laboratory Equipment Safety 14 Emergencies and Accidents 27 Exposure Monitoring and Medical Treatment 29 Training 29 Chemical Safety 31 Understanding Chemical Hazards 31 Material Safety Data Sheets (MSDS) and Labels 38 Laboratory Safety Procedures 42 Chemical Disposal 50 Bio logical S afety 52 Aerosol-Generating Processes 57 Biological Safety Cabinets (Tissue Culture Hoods) 58 BSC Use: Work Practices and Procedures 65 General Biosafety Issues 69 Biological Spills and Decontamination 70 Regulated Medical/Bio-Hazardous Waste Management 71 Summary of Biosafety Levels 73 Summary of Agents 76 Appendices A Example Glove Compatibility Chart 78 B NIOSH Guide on Chemical Storage 79 C Biological Safety Self-Audit Form 83

4 Scope and Applicability

The University of South Dakota is committed to preserving the health and safety of its students, staff and faculty and to protecting the environment and community. It is recognized that the use of hazardous chemicals, potentially pathogenic microorganisms, infectious agents, human tissue and bloodborne pathogens, and/or organisms containing recombinant DNA (rDNA) is necessary in many university research and teaching laboratories. To ensure the safe handling of these agents, the University of South Dakota requires compliance with the NIH Guidelines for Research involving rDNA Materials; BMBL; Occupational Safety and Health Administration (OSHA) standards (i.e., Title 29 CFR Part 1910.1030 and 1910.1450) and with the recommendations provided in the Biological and Chemical Safety Policy.

Responsibilities The Institutional Biosafety Committee (IBC) is charged by the President of The University of South Dakota to formulate policy and procedures related to the use of biohazardous agents and materials. Hazardous biological agents consist of viruses, bacteria, fungi, and non-exempt recombinant DNA organisms listed by the Center for Disease Control and Prevention (CDC) and/or the US Department of Agriculture (USDA). The IBC responsibility does not extend to toxic materials that are not toxins. The Director, Environmental Health and Safety (EHS) is responsible for implementing the University’s radiation, chemical and biological safety programs. In this role, the director functions as the University’s Radiation Safety Officer (RSO), Chemical Hygiene Officer (CHO), and Biological Safety Officer, if the University engages in rDNA research at the Biosafety Level 3). The Director, EHS will: • monitor compliance with The University safety practices and procedures regarding chemical and biohazardous materials • work with faculty and staff to develop and implement appropriate safety practices and policies • ensure that safety audits are preformed periodically and that results are reported to the responsible parties • provide consultation to investigators on matters relating to laboratory safety, appropriate handling and containment of hazardous materials, decontamination and disposal of hazardous wastes • serve as liaison between the University and outside regulatory agencies concerning the use of hazardous materials Ultimate responsibility for the safe conduct of research involving hazardous materials rests with the laboratory supervisor or principal investigator (PI). Each faculty member is responsible for complying with the requirements in this Policy and implementing all necessary precautions to prevent undesirable consequences of experimental work conducted in the laboratory. The PI will: • monitor daily operations of the laboratory • inform / train persons who enter the laboratory and document the training for each person who works in the lab. • ensure that laboratory workers understand and follow the Biological and Chemical Safety Policy and any other laboratory specific safety procedures

5 • maintain a proper inventory and storage of chemicals and biohazardous materials • determine the required level of protective apparel and equipment for a given procedure and insure proper protective equipment is used • insure that laboratory equipment is adequate for any material being ordered and that training is conducted on the hazards associated with the use of any material • establish written operation and decontamination procedures • establish emergency procedures and train all workers on emergency response • insure good housekeeping and chemical / biological hygiene practices are maintained in the laboratory and that it is kept free from clutter and debris • conduct regular laboratory safety and housekeeping inspections, including routine inspections of emergency equipment • report accidents and any other incident involving spill / release, injury, exposure, etc. following University policies • arrange for immunizations and/or health surveillance of laboratory personnel if deemed appropriate for the research project • all design, construction and/or modification of space must be reviewed and approved by the Director of Facilities Management; the USD will not permit renovations that result in unsafe or unhealthy working environments or do not meet national codes While the Director, EHS and the PI are responsible for insuring a safe work site, the laboratory worker is responsible for keeping themselves informed of the risks involved from working in a laboratory. The laboratory worker will:

• follow all safety and health procedures in the Biological and Chemical Safety Policy and any procedures developed by the faculty supervisor in the laboratory • complete required health and safety training sessions. • wear all required personal protective equipment and develop good personal laboratory safety habits • report accidents, injuries and unsafe conditions to the PI or the laboratory supervisor A university is a learning institution, visitors are to be expected. The laboratory supervisor or PI is responsible to insuring the safety of all visitors in the laboratory. High school students destined to work in a laboratory must be trained and their parents must be appraised of the risk and approve, in writing, of the relationship. Children under the age of 18 are prohibited from hazardous materials laboratories unless prior approval has been obtained from the department head and EHS. Children must be supervised at all times by the laboratory supervisor or PI.

6 General Laboratory Practices and Safety Equipment

Today, the attitude of "safety first" permeates the research community, working safely not just something you fall into, it consists of systematically integrating safety procedures into your work with hazardous materials. Here we will review the kernel of general laboratory safety practices, procedures and equipment which all researchers must know, understand and apply to their laboratory work. General Safety Procedures 1. Know the materials you are working with (e.g. chemical, biological, radioactive) a. Refer to and follow your laboratory protocol b. Review the Material Safety Data Sheets (MSDS) for chemicals. These should be available and easily assessable in each laboratory either as hardcopy or electronic. c. Consider the toxicity of materials, the health and safety hazards of each procedure, the knowledge and experience of laboratory personnel and the safety equipment that is available. If you are uncertain about any aspect of handling the material, talk to your PI / supervisor before using. d. For microbiological and biochemical hazards, review Biosafety in the Microbiological and Biomedical Laboratories (BMBL ), 5th Edition The BMBL outlines safety procedures to follow in the laboratory. At this time, no laboratory at USD exceeds Biosafety Level 2. http://www.cdc.gov/biosafety/publications/bmbl5/index.htm 2. Know the location of safety equipment and emergency procedures in your area. 3. Always wear appropriate clothing (e.g. pants, shirts, shoes) and personal protective equipment (e.g. safety glasses, lab coats, gloves) when working in the laboratory. Open-toed shoes and sandals are unacceptable footwear; shorts and skirts are not recommended. These items do not protect the feet and lower legs from spills or broken glass. Remove personal protective clothing and wash before leaving the laboratory. 4. Avoid working alone in the laboratory. When hazardous operations are conducted, arrangements should be made to have another person present in the lab. If you need to work alone in the laboratory (e.g. weekends, holidays, evenings) be sure your PI / supervisor is aware of your intended work schedule. 5. Use a properly operating fume hood when working with hazardous chemicals. Air flows for University fume hoods are verified at least once every two years. You can verify flow direction by using a piece of tissue paper; just insure the tissue does not become trapped in the ex 6. Do not eat, drink, prepare food or apply cosmetics in the laboratory. 7. Keep work areas clean and uncluttered at all times. 8. Do not leave experiments unattended, especially experiments using a heat source such as water baths, hot plates, etc. 9. Unauthorized individuals are prohibited from entering the laboratory. The PI / supervisor should approve all visitors, and visitors / guests should not be left unattended. 10. Persons under 18 years of age should not be involved in procedures involving hazardous chemicals. 11. Volunteer workers in any research laboratory must be registered with Human Resources. 12. Non-laboratory and non-assistance animals are not allowed in campus buildings.

7 Security Laboratory security is an integral part of an effective safety program. To provide a safe working environment, follow the steps outlined below. 1. Keep laboratory doors locked when the lab is unoccupied. 2. Keep stocks of organisms and hazardous chemicals locked during off hours or when the laboratory is unoccupied. 3. Keep an accurate inventory of chemicals, stocks, cultures, project materials, growth media, and those items that support project activities. 4. Notify USD Public Safety (605-677-5342) if you believe materials are missing from laboratory. 5. Inspect all packages arriving at the work area. Do not open suspicious packages. This includes packages from suppliers you are not familiar with and have not ordered materials from, packages not specifically addressed to your laboratory or do not have a return address. 6. When research is completed for the day, ensure that chemicals and biological materials have been stored properly and securely and the general workspace is secure before you leave. 7. Ask strangers (i.e., someone you do not recognize as a co-worker or support staff) to identify themselves and request they leave the laboratory if they are not authorized to be there. Contact USD Public Safety (677-5342) for assistance if you are uncomfortable confronting a stranger. 8. Discuss other security-specific requirements with your laboratory’s PI, supervisor, research staff and graduate students.

Laboratory Safety Equipment All personnel within a laboratory should be familiar with the location of all relevant safety equipment outlined below. Each laboratory should include signage located near each egress door listing the location of each of the following safety equipment. 1. Safety Showers - Safety showers, sometimes called drench showers or deluge showers, are used in an emergency to flush chemicals that laboratory personnel or their clothing have been grossly contaminated with. To use a safety shower, remove any potentially contaminated clothing at the same time you are standing under the shower. The Safety Shower can be used to extinguish a clothing fire, but this is not recommended if the shower is more than a few feet away. The best way to extinguish a clothing fire is to “ Stop, Drop & Roll ,” and then remove clothing. Laboratories with elevated fire risks should also be equipped with a fire blanket that may be used to suppress a fire. Safety showers are maintained and tested annually by Facilities Management. 2. Eye and Face Washes - For chemical splashes of the eye and face, immediately flush with a large quantity of tempered water for 15 minutes. Sinks designated as eye and face wash stations must be equipped with a stay-open valve and should be tested weekly to ensure

8 proper operation and to flush contaminants out of the pipes. Plastic eyewash bottles are not recommended, they do not have enough continuous flow . In general, the emergency eyewash equipment should available within a 10-second walking time from the location of any safety hazard and should not require leaving the laboratory to access. In addition, the path of travel from the hazard to the equipment should be free of obstructions and as straight as possible; remember the victim may not be able to see. 3. Fire Extinguishers - Fire extinguishers are placed in or just outside laboratories depending on the hazards. Facilities Management has performed a basic hazard analysis and determined the type of fire extinguisher needed for each area. For most laboratories, a type ABC fire extinguisher will be available. Facilities Management conducts monthly inspections and annually performs a basic maintenance on all portable fire extinguishers. Any extinguishers that are found to be questionable are immediately replaced. 4. First Aid Kits - First aid kits should be available in each laboratory. Research laboratory, automobile, and office first aid kits can be purchased through normal USD procurement procedures. First aid kits should NOT contain oral medicines (e.g., aspirin), topical creams, liquids or ointments that can cause further discomfort and/or hinder medical treatment. Laboratories are encouraged to check the contents of the first aid kits and replace materials past the expiration date on the item or in need of replenishment. 5. Laboratory Safety Information - MSDS, emergency procedures, safety policies and other references should be readily available for all laboratory personnel. The laboratory should designate a single location for this information. It is also recommended that copies of the material be stored in a second location, just in case primary copy is lost, damaged or misplaced. 6. Door Postings and Other Signs - Hazard and emergency information signs may be posted on the laboratory door or wall facing the corridor. These signs are used by emergency response personnel to identify hazards within the laboratory. A listing is maintained by USD Public Safety detailing the responsible faculty member and other persons to be contacted in the event of an emergency. This listing is reviewed every six months to ensure accuracy. It is needed to provide a point-of-contact in the event of an accident, chemical spill, fire or personal injury. 7. Open Floor Drains and Sink Traps – sometimes a sink trap or drain dries up. To reduce odors in buildings, sink traps and floor drains should be flushed at least weekly with one or two liters of water. Sinks traps and floor drains located in areas where temperature may drop below freezing should be flushed with biodegradable antifreeze approved by USD Facilities Management. Laboratory spaces that are not used for long periods of time should be checked regularly to assure that floor drains and sink traps are filled with water. Do not place any equipment over floor drains or sinks which can obstruct this routine maintenance. 8. Sharp Containers and Glass Only Boxes - Sharps containers are used for the disposal of hypodermic needles and syringes, razor blades and other sharp items. When ¾ full, sharps containers should be sealed and processed according to local unit procedures. While sharps containers may be used for contaminated broken glass, never “rebreak” such glass to facilitate placement in a sharps container. If the contaminated, broken glass too large to fit in the normal sharps container, a “broken glass” box may be used to contain the glass. This contaminated glass box should be labeled and handled in a manner consistent with the contamination. “Glass Only” boxes are used for the disposal of “clean,” broken glass only. When ¾ full, the boxes should be sealed, labeled “Broken Glass” and disposed in a dumpster. Remember, broken glass that is biologically or chemically contaminated must be handled as a contaminated waste.

9 9. Mechanical Pipetting Aids - Mechanical pipetting devices should be used. Mouth pipetting is unacceptable. 10. Vacuum Line Filtration/Air Line Filtration - To prevent fluid and aerosol contamination of the central or local vacuum/air system in certain buildings, it is recommended that a particulate air filter trap (e.g., high efficiency particulate filter cartridge) be placed in any suction/air tubing immediately before the main control valves. These filters protect the system from corrosion, rust, etc., and should be replaced annually or when there is evidence of filter blockage, failure or wetness.

Laboratory Safety Surveys The Director, Environmental Health & Safety (EHS) conducts a safety survey each year. The results of the laboratory survey are discussed with the PI / supervisor and other assigned workers. The safety inspection includes but is not limited to: fume hood operation, laboratory techniques, emergency and safety equipment, chemical storage, electrical safety, general housekeeping, and radioactive and biohazardous material storage. The PI / supervisor is responsible for correcting operational deficiencies in a reasonable time. Facility related deficiencies (e.g., inoperable safety shower, inadequate fire extinguisher) are directed to Facilities Management for review and resolution. More frequent laboratory safety surveys may be conducted in laboratories with particularly hazardous substances or biological hazards. Personal Protective Equipment (PPE)

Personal protective equipment (i.e., eyewear, goggles, gloves, lab coat, etc.) help protect the worker from both physical effects of a chemical or from internal deposition. This PPE must be available for laboratory personnel who are working with hazardous materials. Laboratories must also provide personal protective equipment (i.e. safety glasses, laboratory coat) for visitors. In areas where eye or face protection is needed, a sign indicting what level of personal protective equipment is required must be posted in a prominent location.

Eye and Face Protection Eye and face protection must be worn in the laboratory when there is a potential for contact with hazardous chemicals or other agents (e.g. UV or laser radiation, biohazardous materials, aerosolized material, flying objects.). The type of protection needed depends on the hazard (e.g. chemical, UV, laser, impact). Remember that procedures that may promote aerosolization or volatilization of materials should always be regarded as potentially high risk. As a rule, whenever laboratory chemicals are used, appropriate eye protection is mandatory and chemical splash goggles recommended. Goggles over eyeglasses or prescription safety glasses with side shields should be worn. Ordinary prescription glasses and some ‘safety’ glasses do not meet these standards, especially if a splash hazard exists. Face shields are worn when working with an agent that may adversely affect the skin on the face and/or when proper eye protection is not enough. Eye, skin and face protection are required when working with corrosive or reactive chemicals, with glassware under pressures, in combustion

10 and other extreme temperature operations and whenever there is a possibility of an explosion or implosion, no matter how remote the possibility. Special safety glasses and face shields may also be required for work with UV light and laser radiation which is absorbed by the eyes or skin (chemical splash goggles are not adequate for these types of work).

Laboratory Coats, Gloves and Other Protective Clothing Laboratory coats and shoes should be worn when working in the laboratory. The shoe should fully cover the foot and open toed-shoes, sandals, flip-flops, clogs, etc. are unacceptable for laboratory work. The laboratory coat should be made of a material suitable for the research (e.g., polyester lab coats may not be appropriate in laboratories using organic solvents or open flames). Cotton or cotton/polyester blend lab coats may retain liquid contaminations and may pose a cumulative exposure risk. Lab coats should be regularly cleaned following USD procedures. Depending on the type of work, additional personal protective equipment, such as gloves and aprons may be necessary. Lab coats, aprons and gloves should be removed when leaving the laboratory. Lab coats, aprons and gloves should be made of a material rated for the hazard likely to be encounter in the laboratory. Remember, no material should be regarded as fully protective. Lab coats, aprons and gloves should be removed immediately if they are contaminated or torn. In situations involving extremely hazardous materials, disposable aprons and double gloves are recommended. Protective gloves should be carefully selected for their degradation and permeation characteristics so they will provide proper protection. The thin, latex, vinyl or nitrile gloves, popular for their dexterity are not appropriate for most laboratory uses. These provide limited permeation protection and are easily torn. When working with hazardous chemicals, consult the chemical compatibility chart provided in most supplier catalogs or web sites (e.g., http://www.ansellpro.com/download/Ansell_8thEditionChemicalResistanceGuide.pdf ) to help select the proper gloves and other protective clothing. An example glove chart is at Appendix A. Personal protective equipment should not be worn outside of the laboratory setting unless necessary for the research.

Respiratory Protection The use of air-purifying respirators or power face masks for laboratory work is discouraged. In general, respirators should not be used in laboratories because they protect only the wearer and may lead to a sense of false security and, unfortunately, often to sloppy technique and carelessness. There are specific requirements for respirator use. They may require periodic calibration and monitoring, workers need specific training and fit testing before the mask can be worn effectively, there may also be the need for a physical evaluation. This is also true for the surgical facemask and the popular N96 mask. Properly operating laboratory fume hoods and

11 biosafety cabinets provide the best overall protection from chemical and biological hazards in the laboratory.

Protective Clothing Outside the Laboratory The use of appropriate gloves, safety glasses, lab coats and other personal protective equipment within the laboratory is designed to provide for a healthy and safe working environment. Wearing personal protection equipment outside of the laboratory is regarded as poor laboratory practice. If the PPE is contaminated, then the wearing of the PPE outside of the laboratory increases the risk of spreading the contaminant to other non-research areas. If you are transporting something from one lab to another, follow these guidelines: • Wearing gloves outside the lab should be minimized, except to move hazardous materials between laboratories. Instead, transport chemicals from place to place on a cart, in a clean secondary container, or in a bottle carrier with secure handles. Be careful not to overload transport systems capacity. • If there is a need to transport hazardous materials, use a clean, ungloved hand to touch common surfaces and a gloved hand to carry the items: the one-glove rule. Alternatively, package the material so it may be handled without gloves. • Gloves should never come in contact with door handles, elevator buttons, telephones, lavatory faucets, vending machines, water fountains, keyboards, ice making machines, or other surfaces outside the laboratory. • For the sake of safety, appearances, and courtesy, do not wear contaminated, stained, or potentially contaminated lab coats and other research clothing and equipment outside of the laboratory. • Do not carry specimen Dewars or covered, polystyrene boxes with dry ice or cryogenic liquid in a private vehicle. Be aware that strict federal, state and local regulations address the transport of hazardous (biological, chemical, radiological) materials on public roads. Fume Hoods and Biosafety Cabinets

All work with hazardous materials must be conducted in the appropriate fume hood or biological safety cabinet. General room ventilation, including open windows, will not provide adequate ventilation and protection for benchtop work which produces hazardous gases, vapors, dusts and aerosols; these may actually spread the contamination over large areas. All work with corrosive, flammable, odoriferous, toxic or other dangerous materials shall be conducted only in a properly operating chemical fume hood rated for that material. Ductless fume hoods are not acceptable unless specifically reviewed and approved by the Director, EHS. Certain biological agents will require the use of certified biological safety cabinets (BSC). When it is not possible to use a BSC, the University Biosafety Committee will review the project to determine if work can be conducted safely at USD.

12 Fume Hoods Fume hoods are checked annually for adequate flow. The velocity of the air at the face of the hood is measured with the sash set to the appropriate test opening and the results are posted on a sticker, which is attached to the upper right-hand corner of the sash. Variable air volume (VAV) hoods maintain a constant face velocity at different sash heights. Generally, when conducting experiments, researchers should have the sash closed as much as possible. Hoods that do not meet the minimum exhaust requirements during inspections are posted with “FAILED ”. A “FAILED” fume hood should NOT be used until repairs have been made and the hood is certified as meeting minimal specifications.

Hoods should NOT be used for chemical or hazardous waste storage. The EPA prohibits intentional hazardous air emissions. Waste containers should be tightly capped when not being used. If the hood is full of chemical containers, flow will be disrupted and the user may be reluctant to clean out the hood and may be at risk for exposure or injury when cleaning out the hood.

Procedures for Proper Use of Fume Hoods Before using a hood, make sure air is entering the hood, that the hood is functioning properly and the hood is rated for the procedure. Some hoods are specifically rated for materials such as perchloric acid and some research gases and should not be used for other procedures for safety reasons. Report any problems to the PI / supervisor. NEVER obstruct baffle openings or place bulky items in the hood that will potentially interfere with the normal operation. Also, remember that drafts from open fans, air conditioners, windows and doors, traffic near the hood face, and even your own sudden movements will disrupt the airflow patterns in the fume hood and potentially compromise the hoods operation and your safety. Before each use, be sure to verify the following: • air is entering the unit • baffle openings are not blocked and air is flowing properly • you are able to conduct all work at least six inches from the edge of the hood • the sash is lowered to provide optimal protection to you while not disrupting the overall operation of the hood

13 • the hood is clean and uncluttered, wipe up spills immediately • be aware that drafts from open windows, open doors, fans, air conditioners, high traffic walkways may interfere with normal hood exhaust

Fume Hood Alarms Fume hood alarms indicate substandard operation of fume hoods. They are installed on every new fume hood system. The fume hood alarm (audio/visual) will indicate an exhaust flow malfunction. If the fume hood alarm sounds, close the sash to the minimal level and immediately notify the PI / supervisor. They should initiate the repair process and label the hood with a “Do Not Use” sign.

Biological Safety Cabinets Class II (vertical laminar flow) biological safety cabinets (BSC) provide a partial containment system for the safe handling of pathogenic microorganisms. BSCs are NOT fume hoods and should not be used with toxic chemicals. BSCs that recirculate air should not be used for procedures requiring special gases. To ensure safety, BSCs must be used correctly with good microbiological techniques and be in proper mechanical working order. BSCs are certified for performance on installation and annually thereafter. BSCs moved to a new location should not be used until certified for use. Because most BSC hoods are shared by several laboratories, the BSC must be decontaminated after each use. The ultraviolet light should be routinely checked and replaced on a regular schedule to ensure optimal function.

Laminar Flow Hoods Laminar flow hoods are present in a number of lab facilities. This type of hood provides a clean work environment by drawing in air and filtering it before exhausting it across the work surface toward the front of the hood. Thus, laminar flow hoods should never be used with any hazardous material because they exhaust air toward the operator. Similarly, materials considered toxic, infectious or sensitizing, including volatile chemicals, cell culture materials (except plant cell cultures) or drug formulations should not be used in laminar flow hoods. Laboratory Equipment Safety Remember the proverb, "familiarity breeds contempt?" Working within your lab day-after-day without any safety incidents may cause a worker to forget the potentially hazardous nature of

14 their laboratory environment. Lab instruments and equipment that operate at high voltage, high pressures, high speeds or high temperatures, are potentially dangerous. Such equipment should be inspected and maintained regularly and serviced to reduce the hazards from failure. Many of the pieces of equipment you use daily can pose a hazard. When using any equipment remember:
use the correct equipment
know how to operate the equipment
inspect the equipment
use the equipment properly Equipment purchased for use in your lab is designed to be safely operated. Use the equipment for its intended purpose only. Do not modify or adapt equipment without guidance from the equipment manufacturer. Do not defeat, remove, or override equipment safety devices. Be aware of the hazards of each type of equipment. Before using a new piece of equipment, or one you have not used before, review the manufacturer’s literature, becoming familiar with how to operate the equipment and how to apply appropriate safeguards. Always inspect equipment before using and keep the area around instruments and equipment clear of obstructing materials. Another potential hazard from operating equipment is aerosol production. An aerosol refers to the physical state of liquid or solid particles suspended in air. Aerosols containing infectious

agents and hazardous materials can pose a serious risk because: • if inhaled, small aerosol particles can penetrate and remain deep in the respiratory tract, • aerosols may remain suspended in the air for long periods of time, and • aerosol particles can easily contaminate equipment, ventilation systems, and skin. Types of operations which may lead to the production of aerosols include: − centrifuge − blender − shaker − magnetic stirrer − sonicator − pipet − vortex mixer − syringe and needle − vacuum-sealed ampoule − grinder, mortar and pestle To reduce or eliminate the hazards associated with aerosols: • Conduct procedures that may produce aerosols in a biological safety cabinet or a chemical fume hood. • Keep tubes stoppered when vortexing or centrifuging. • Allow aerosols to settle for one to five minutes before opening a centrifuge, blender or tube. • Place a cloth soaked with disinfectant over the work surface to kill any biohazardous agents. • Slowly reconstitute or dilute the contents of an ampoule. • When combining liquids, discharge the secondary material down the side of the container or as close to the surface of the primary liquid as possible. • Avoid splattering by allowing inoculating loops or needles to cool before touching biological specimens. • Use a mechanical pipetting device.

15 Centrifuges Centrifuging presents the possibility of two serious hazards: mechanical failure and aerosols. While the most common hazard associated with centrifuges is a broken tube, given the high speeds achieved and the necessity to maintain balance it is important to properly load the centrifuge, operate it only a speeds recommended by the manufacturer, wait until it has completely stopped before removing all of the sample and properly clean the unit. The following guidelines help reduce accidents: • When loading the rotor, examine the tubes for signs of stress and discard any tubes that are damaged. • Inspect the inside of each tube cavity or bucket; remove any glass or other debris from the rubber cushion. • Insure that the centrifuge has adequate shielding to guard against accidental flyaways. • Use a centrifuge only if it has an interlock that deactivates the rotor when the lid is opened. • Do not overfill a centrifuge tube to the point where the rim, cap, or cotton plug becomes wet. • Always keep the lid closed during operation and shut down and do not open the lid until the rotor is completely stopped. • Do not brake the head rotation by hand. • Do not use aluminum foil to cap a centrifuge tube, it can rupture or detach. • When balancing the rotors, consider the tubes, buckets, adapters, inserts, and any added solution. • Stop the rotor and discontinue operation if you notice anything abnormal such as noise or vibration. • Rotor heads, buckets, adapters, tubes and plastic inserts must match. Some low-speed and small portable centrifuges may not have aerosol-tight chambers and may allow aerosols to escape. In these instances, use a safety bucket to prevent aerosols from escaping. High-speed centrifuges pose additional hazards due to the higher stress and force applied to their rotors and tubes. For high-speed centrifuges:
Filter the air exhausted from the vacuum lines
Keep a record of rotor usage to reduce the hazard of metal fatigue
Frequently inspect, clean, and dry rotors to prevent corrosion or other damage. Proper care and inspection of centrifuge rotors is important. Periodic cleaning of the rotor and chamber is necessary to keep the unit in proper working order. Clean any spills immediately. Other tasks which can assure safe use of centrifuges include: • Review the rotor SOP to insure it includes the manufacturer's safety instructions. • Visually inspect the rotor for mechanical or chemical damage prior to each use. Inspect the underside of the rotor, the web area of the rotor and the outer rim. Insure the top and bottom pieces of the rotor are tightly connected. • Certain chemicals (e.g., phenol) attack plastic rotors and some nucleic acid extraction kits can damage the rotor. Chemical damage often appears as discoloration, crazing, granulation, peeling or similar deterioration of the rotor finish.

16 • Mechanical damage such as cracks, scratches, or gouges can often be seen or detected as an increase in noise or vibration during a spin. Do not ignore excessive vibration that does not resolve after rebalancing and checking the fit of the rotor cover. Do not use the rotor if any damage or change is evident. • Always use the rotor cover. Use an aerosol seal for containment of pathogenic materials. If you see scoring around the circumference of the top of the plastic rotor cover, replace the rotor, since this may indicate rotor deformation

Compressed Gas Cylinders Compressed gases in the lab present chemical and physical hazards. If compressed gas cylinders are handled incorrectly, they can be lethal. A broken cylinder valve can cause a cylinder to act like a rocket. Exposure to some gases, such as hydrogen sulfide from a cylinder leak, can be lethal. Flammable gases pose a double hazard. Restrict the use of flammable gases (e.g., hydrogen, acetylene, propane, butane, etc.) to no more than three 220-ft 3 cylinders of flammable gases or oxygen per 500 ft 2 of unsprinklered laboratory space and use no more than 400 ft 3 of hydrogen gas in areas below ground level. If possible, use flammable gases only in areas where a flammable gas detection system is installed. Do not flare or burn off residual gas from laboratory equipment, duct the gas to a scrubber and neutralize it. Here are some other precautions for use of cylinders: • Only use regulators approved for the type of gas in the cylinder. Do not use adapters to interchange regulators and never use improvised adapters. • Always wear impact resistant glasses or goggles when working with compressed gasses. • Never refill gas cylinders. • Limit the amount of highly reactive, toxic, or flammable chemicals to the quantity necessary for planned experiments, or that will be used within a few months. Avoid the use of these compounds whenever possible • Before using, check all connections under pressure for leaks. Swab connections with a soap solution and look for bubbles. • Don't leave regulators and valves on corrosive gas cylinders except when they are in frequent use. Work the valve stem of a corrosive gas cylinder often to keep it from freezing. • Do not force valve stems; they can easily snap off. • Turn off both the main valve and regulator when not using the cylinder. • Do not use copper (> 65% copper) connectors or tubing with acetylene, the acetylene can form explosive compounds with silver, copper and mercury. • Clearly mark empty cylinders "empty." Secure empty cylinders in your building's designated cylinder storage area until they are picked up by the vendor. Lecture bottles are small gas containers that can become a serious disposal problem for the University. This high disposal cost (e.g., $2,000 per lecture bottle) reflects the hazards of laboratory gases and the difficulty of disposing of lecture bottles. Exotic and toxic gases (e.g., arsine, phosgene and nitrogen dioxide) are often supplied in lecture bottles. The most expensive to dispose of are lecture bottles that are old, have inoperable

17 valves or have no markings to indicate the contents. Old lecture bottles can leak or spontaneously rupture. Here are some steps to take to minimize the hazards and cost of lecture bottle disposal. Store your cylinders safely. Lecture bottles should be stored in a separate ventilated cabinet where the temperatures do not exceed normal room temperatures. Because lecture bottles may contain gases that are liquefied at pressures below the 150 atmospheric limit, they can be more susceptible to increased pressure with heating; especially if critical temperature is attainable near room temperatures, lay them on their sides with their valves pointed toward the ventilation port. Do not store corrosives with lecture bottles. The corrosive vapors of chemicals such as hydrochloric acid or nitric acid can destroy markings and damage valves. Other considerations for the storage of large (50 inch) compressed gas cylinders: • Buildings where large gas cylinders are used should have a designated area for storing newly received cylinders and empty cylinders for return to the vendor. Use chains or straps to keep cylinders secured to a wall or a cylinder rack. • Never accept a cylinder if the name of the contents is missing or illegible. • Don't rely on color codes for identification. Don't accept a cylinder if it only has a color code or the color code does not match the printed name. • Transport cylinders on a cylinder cart with a safety chain. Never move a gas cylinder unless the cylinder cap is in place. Do not move a cylinder by rolling it on its base. Do not lift cylinders by the cap. • Always secure gas cylinders to a wall or a stable bench with clamps, straps or chains. Fasten cylinders individually, not ganged, and in a well ventilated area. Even empty cylinders must be secured. Bench clamps are available from chemical suppliers. • Don't remove protective cylinder caps until the cylinder is secured. Replace cylinder caps before returning cylinders to their storage area. • A maximum of 3 flammable, oxygen or health hazard gas cylinders should be stored per 500 square feet of area. • Store cylinders of flammable and oxidizing agents at least 20 feet apart. • Keep only the cylinders that are necessary for current work in your lab. • Keep cylinders away from all sources of heat, sparks, flames and direct sunlight to prevent pressure increases. Do not heat cylinders to raise internal pressure. • Do not store gas cylinders in hallways or public areas. Track their use. Attach a clipboard to the cabinet or a tag to the cylinder to record dates and the weight of bottles before and after use. Don't mark a bottle "empty" unless you know that it is actually empty. Buy what you need; use what you buy. Buy only the amount of gas necessary for your work and use it all. Consider the efficiency of sharing gases with other labs.

18 Annually inspect your lecture bottles and cylinders. Examine your lecture bottles and cylinders for the integrity of their markings, tare weight tags and for corrosion. Use a soap solution to check for leaks at the valves. If cylinder label and valve tag do not agree or if there is any question as to the contents, do not use. Dispose of all lecture bottles that you have no plans to use in the immediate future. For larger gas cylinders, there is usually a charge for the time the cylinder is in service. Often this charge is in the form of demurrage, that is, a rental charge that starts after an agreed upon period of time. These charges are quite small on a monthly basis, but can add up over time. For example, a scientist on campus used a cylinder of nitrogen from 1986 to 1992. The price of the gas was $8.82 but he paid over $135 in cylinder rental. To minimize these rental charges:
Order only the quantities of the gas that you need.
Keep track of the location of each cylinder and the date you received it.
Use your cylinders on a first-in first-out basis.
If you have no plans to use a cylinder for several months, it may be worthwhile to return a partially full cylinder rather than storing it. Return unwanted or surplus cylinders or lecture bottles to the vendor. Some vendors will take back surplus gas and empty lecture bottles. When buying gases, ask the vendor about providing return service. Consider the high cost of disposal if the vendor will not accept surplus gases for return. Safety can help you with packaging, labeling and shipping cylinders to be returned.

Cryogenic Liquids Cryogenic liquids are hazardous because of the physical and chemical characteristics of their super-cooled state. Cryogenic liquids may cause explosions, fires, asphyxiation, tissue destruction or embrittlement of structural materials.

Follow these guidelines for using cryogenic liquids: • Avoid skin and eye contact with cryogenic liquids. Always wear eye protection, preferably a face shield. Don't use gloves that can be frozen to the skin. • Keep cryogenic liquids away from all sources of ignition. • Store cryogenic liquids in a well-ventilated area to avoid buildup of flammable gases or the displacement of air. Do not inhale cryogenic vapors. • Store cryogenic liquids in double-walled, insulated containers (e.g., Dewar flasks). Handle Dewar flasks carefully. Tape them thoroughly to prevent the release of a large number of tiny glass slivers in the event the flask

shatters. • Pre-cool receiving vessels to avoid thermal shock and splashing. • Select work materials wisely. Cryogenic liquids alter the physical characteristics of some materials. Accidents have been reported where Pyrex tubes have failed causing injury. • Use tongs to place and remove items in cryogenic liquid. Rubber and plastic may become very brittle in extreme cold; handle these items carefully when removing them from cryogenic liquids.

19 • Be careful when transporting cryogenic containers; use a cart for large cryogenic containers.

Glassware Use Broken glass is one of the most common causes of laboratory injuries. To reduce the chance of cuts or punctures, be careful when working with glassware. Inspect glassware for chips and cracks before use. When you cut glass, use hand protection and fire-polish all cut surfaces. These additional tips will help to reduce the risk of injury: • Never use laboratory glassware to serve food or drinks. • Use care in handling and storing glassware to avoid damaging it. • Discard or repair any chipped or cracked items. • Leave an air space of at least 10% in containers with positive closures. • When possible, substitute plastic or metal connectors for glass connectors. • Thoroughly clean and decontaminate glassware after each use. • When inserting glass tubing into rubber stoppers, corks, or tubing, use adequate hand protection (e.g., gloves or a towel), lubricate the tubing and hold hands close together to minimize movement if the glass breaks. • Use thick-walled, round-bottomed glassware for vacuum operations. Flat-bottomed glassware is not as strong as round-bottomed glassware. Carefully handle vacuum-jacketed glassware to prevent implosions. Dewar flasks, vacuum desiccators, and other evacuated equipment should be taped or shielded and for vacuum work, use only glassware designed for that purpose. • Large glass containers are highly susceptible to thermal shock. Heat / cool large glass containers slowly. Use Pyrex or heat-treated glass for heating operations. With proper precautions, work with glassware can be conducted safely. These additional handling precautions will help reduce the risk of injury.
When handling cool flasks, grasp the neck with one hand and support the bottom with the other hand.
Lift cool beakers by grasping the sides just below the rim. For large beakers, use two hands, one on the side and one supporting the bottom.
Never carry bottles by their necks.
Use a cart to transport large bottles of dense liquid. Regardless of the precautions you take, glass may still break. Broken glass poses a hazard for puncture wounds and injection of hazardous chemicals. • Do not pick up broken glass with bare or unprotected hands. Use a brush and dustpan to clean up broken glass. Remove broken glass in sinks by suing tongs for large pieces and cotton held by tongs for small pieces and slivers. • Glass contaminated with biological, chemical or radioactive material must be decontaminated before disposal. • Dispose of broken glass and other sharps according to University policy. Frozen Glass Stoppers. Ground glass stoppers frozen by contact with base solutions may be welded hopelessly. Those welded by fluoride solutions may have built-up pressure of silicon tetrafluoride. Be careful when removing frozen glass stoppers. First, try soaking the stopper in hot water to expand the glass. If this doesn't work, try a special solution for freeing frozen joints:

20 − 10 parts chloral hydrate, − 5 parts glycerin, − 5 parts water and − 3 parts concentrated HCl Paint the solution on the frozen ground glass joint or immerse into the solution. If you need to remove a stopper by tapping, wrap the stopper in a cloth or paper towel and wear gloves to protect your hands and prevent injury in case of breakage.

Electrical Safety Laboratory fires can often be started by the careless handling of electrical equipment. Labs have stills, water baths and other apparatus that can overheat or cause electrical shocks. Minimize electrical safety hazards with the following:
Before use, check all electrical apparatus for worn or defective insulation and loose or broken connections. Power cords should be checked closely and replaced if defective.
Connect all ground wires to clean metal, avoid painted surfaces. Use three-prong grounded plugs whenever possible.
Keep electrical wires away from hot surfaces.
Don't allow water and other potentially destructive liquids to leak on electrical wires, switches and outlets.
Avoid the use of extension cords. University policy only allows extension cords in temporary situations and the cord must be grounded.
Never touch a switch, outlet or other electric power source with wet hands.
Don't use homemade or makeshift wiring, call an electrician for wiring. Electrophoresis equipment is an example of a system which may be a major source of electrical hazard. This is because there is both high voltage and conductive fluid which presents a potentially lethal combination. Workers may be unaware of the hazards associated with electrophoresis. A standard electrophoresis operating at 100 volts can deliver a lethal shock at 25 milliamps. Even a slight leak in the device tank can result in a serious shock. Besides the above, use these safety precautions to protect yourself from the hazards of electrophoresis and electrical shock. • Follow the manufacturer’s operating instructions. • Use physical barriers to prevent inadvertent contact with the apparatus. • Use electrical interlocks. • Do not disable safety devices. • Use warning signs to alert others of the potential electrical hazard. • Frequently check the physical integrity of the electrophoresis equipment. • Turn the power off before connecting the electrical leads, opening the lid or reaching into the chamber. • Use only insulated lead connectors and connect one lead at a time using one hand only. Insure that your hands are dry when connecting the leads. • Keep the apparatus away from water and water sources.

21 Heating Systems Next to glass failure, the most common source of laboratory injury is the improper manipulation of heating apparatus, particularly gas burners. These systems are needed to provide the heat needed to effect a reaction or a separation. These include: − open flame burners − oil and air baths − furnaces − hot plates − hot air guns − ashing systems − heating mantles − ovens − microwave ovens Some laboratory heating procedures involve an open flame. Common hazards associated with laboratory heating devices include electrical hazards, fire hazards and hot surfaces. When temperatures of 100 o1C (212 oF) or less are required, it is safer to use a steam-heated device than an electrically heated device because they do not present a shock or spark hazard and they can be left unattended with the assurance that their temperature will never exceed 100 oC (212 oF). Follow these guidelines when using heating devices: • Before using any electrical heating device insure that the heating unit has an automatic shutoff to protect against overheating and that all connecting components are in good working condition. • Heated chemicals can cause more physical damage much more quickly than the same chemical would cause at a lower temperature. • Heating baths should be equipped with timers to insure that they turn on and off at appropriate times. • Use a chemical fume hood when heating flammable or combustible solvents. Arrange the equipment so that escaping vapors do not contact heated or sparking surfaces. • Use non-asbestos thermal-heat resistant gloves to handle heated materials and equipment. • Do not leave oil baths unattended. Place your oil bath within a plastic or metal tray to contain any spills. • Perchloric acid digestions must be conducted in a perchloric fume hood. • Minimize the use of open flames. • It is a good idea to connect all exit ports from gas chromatographs (GC), atomic absorption (AA) spectrometers and other analytical instruments to an exhaust ventilation system to exhaust toxic contaminants from the laboratory. Generally speaking, emission from a GC is not significant, but an AA that uses a flame should be installed with a chimney anyway. Pressurized Systems Processes requiring high pressures present a physical hazard should the equipment fail. High pressure operations should only be conducted in equipment specifically designed for this use and only by persons trained to use the equipment. Do not conduct a reaction in, or apply heat to, a closed system apparatus unless the equipment is designed and tested to withstand pressure (e.g., such systems may be stamped with safe operating pressures or have a plate attached with the information). Pressure systems should also have an appropriate relief valve. Additionally, pressurized systems must be fully shielded and should not be conducted in an occupied space until safe operation has been assured. Until safe operation is assured, remote operation is mandated. Points to remember for pressurized work: • Minimize risk and exposure.

22 • Identify and assess all hazards and consequences. In particular, consider how failures may take place and their consequences. • Use remote manipulations whenever possible and conduct the procedure with equipment at locations remote from personnel. • Minimize pressure, volume and temperature. The stored energy available for release is proportional to the total volume and pressure. • Design conservatively. Don't assume that the apparatus will have an "inherent safety factor," sometimes this supposed factor will not be present. • Use material with a predictably safe failure mode. Seek material which will fail in a ductile manner. Do not use a brittle material in an occupied area unless it is properly shielded or barricaded. Remember, a failure is possible if the material is not correct for the application, if fabrication is haphazard, or the right process is not employed. • Demonstrate structural integrity by a proof test. Ensure that the components of the pressurized system will maintain structural integrity at the maximum allowable working pressure. • Operate within the original design parameters. Do not exceed maximum allowable working pressure (MAWP). Do not change working fluids or service environments without considering the consequences of a failure. • Provide backup protection. Suitable pressure relief valves should be installed to insure that the pressure level will stay within safe limits if the equipment malfunctions of is improperly operated. • Use quality hardware. When buying material, insist on information about the manufacturer's design, test and quality control. • Use protective shields / enclosures. These are essential if quality hardware is not procured and they provide insurance for the unpredictable failure. • Use tie-downs to secure tubing, hoses, and piping. Remember, if a line fails under pressure it can whip about unless it is restrained. • Do not leave a pressurized system unattended.

Vacuum Systems Vacuum systems may have similar hazards as high pressure work. Here, vacuum lines and other glassware at sub-ambient pressure may implode. Flying glass is not the only hazard. Dangers associated with possible toxic chemicals contained in the system as well as fire (e.g., of a solvent

stored in the container). Precautions to help insure safety: • Insure that pumps have belt guards in place during operation. • Insure that service cords and switches are free from defect. • Always use a trap on vacuum lines to prevent liquids from being drawn into the pump, house vacuum line, or water drain. • Replace and properly dispose of vacuum pump oil that is contaminated with condensate. • Place a pan under pumps to catch oil drips. • Do not operate pumps near containers of flammable chemicals.

23 • Do not place pumps in an enclosed, unventilated cabinet. Glassware used in vacuum operations may pose a hazard if it breaks. To reduce the injury from glass debris: • Only heavy-walled round-bottomed glassware should be used for vacuum operations. The only exception is glassware specifically designed for vacuum operations (e.g., Erlenmeyer filtration flask). • Wrap exposed glass with tape to prevent flying glass if an implosion occurs. • Carefully inspect vacuum glassware before and after each use. Discard any glass that is chipped, scratched, broken, or otherwise stressed. Glass desiccators may develop a slight vacuum due to contents cooling. When possible, use molded plastic desiccators with high tensile strength. For glass desiccators, use a perforated metal desiccator guard. Vacuum pumps often have a cold trap in place to prevent volatile compounds from getting into hot pump oil and vaporizing into the atmosphere and to prevent moisture contamination in a vacuum line. Guidelines for using a cold trap include: • Locate the cold trap between the system and vacuum pump. • Insure the cold trap is big enough and cold enough to condense vapors present in the system. • Check frequently for blockages in the cold trap. • Use isopropanol/dry ice or ethanol/dry ice instead of acetone/dry ice to create a cold trap. Isopropanol and ethanol are cheaper, less toxic, and less prone to foam. • Do not use dry ice or liquefied gas refrigerant bath as a closed system; these can create uncontrolled and dangerously high pressures.

Distillation of Organic Solvents Potential hazards from distillations arise from pressure buildup, flammable materials, and the use of heat to vaporize the chemicals involved. Use care construction of the system to insure effective separation and to avoid leaks which could lead to fires or contamination. Take precautions with distillations and reactions, especially when they run overnight. Use these guidelines for safe distillations:
Prevent overheating by ensuring that all hoses and connections are securely tightened.
Always leave a phone number where you can be reached. Post it on the door of your lab so that emergency responders can contact you for information in case of a fire or emergency.
Use boiling chips or stir bars to prevent bumping during distillations, refluxing, and similar procedures.
Be aware when distilling chemicals that certain types may auto-oxidize and accumulate peroxides. Peroxides can explode when heated and concentrated during a distillation.
Use only round-bottom flasks for vacuum distillations. Erlenmeyer flasks are more likely to implode. Vacuum distillations or evaporations should always be shielded in case of implosion.

Refrigerators / Freezers Using a household refrigerator to store laboratory chemicals is hazardous for several reasons. Many flammable solvents are still volatile at refrigerator temperatures. Refrigerator temperatures are typically higher than the flash point of most flammable liquids. Additionally,

24 the storage compartment of a household refrigerator contains numerous ignition sources including thermostats, light switches and heater strips. Furthermore, the compressor and electrical circuits, located at the bottom of the unit where chemical vapors are likely to accumulate, are not sealed. Laboratory-safe and explosion-proof refrigerators typically provide adequate protection for chemical storage in the laboratory. For example, laboratory-safe refrigerators are specifically designed for use with flammables since the sparking components are located on the exterior of the refrigerator. Explosion-proof refrigerators are required in areas that may contain high levels of flammable vapors (e.g., chemical storage rooms). These guidelines will help insure safety: • Never store flammable chemicals in a household refrigerator. • Do not store food or drink in a laboratory refrigerator / freezer. • Insure that all refrigerators are clearly labeled to indicate suitable usage. • Laboratory-safe and explosion-proof refrigerators are identified by a manufacturer label. • Refrigerators used to hold food should be labeled, " For Food Only ."

Autoclave Safety Autoclaves are used in many areas to sterilize materials by high heat and pressure. The hot (132 oC [270 oF]), pressurized (30 psi) steam that autoclaves generate make them serious burn hazards. Burns can result from physical contact with the autoclave structure and from contact with the steam leaving the unit. Explosive breakage of glass vessels due to temperature stresses can produce mechanical injury, cuts and burns during opening and unloading the unit. Burns can also result from careless handling of vessels containing hot liquids. Additionally, because of the extreme conditions created inside steam autoclaves, they can easily malfunction if not carefully maintained. Because each autoclave make / model has unique characteristics, it is imperative that you read and thoroughly understand the manufacturers operating procedures before you use an autoclave for the first time. An autoclave uses different patterns of high heat, vacuum and pressure to sterilize material. The main types of runs are: − liquids, for any type of water-based solutions, − dry goods with vacuum, and − dry goods without vacuum

25 Autoclaves often have an additional drying cycle in which hot air is drawn through the chamber to dry materials after sterilization. Remember, controls for different brand of autoclave vary making it important to carefully follow the manufacturer's instructions about loading, load sizes, and cycle types and settings. The liquids run is longer than the other two types, but uses lower temperatures to minimize evaporation of the liquids being sterilized. Insure seals on liquid containers are loose so any expanding vapors produced during heating will not cause an explosion. Use a tray with a solid bottom and walls to contain the bottles and catch spills. Never autoclave any flammable or volatile liquids. The dry goods with vacuum run moves steam and heat into the deepest part of large bags or bundles of materials and produces the best conditions for killing persistent organisms. During this procedure, they chamber alternates between cycles of high pressure, steam and vacuum. It is important that steam and pressure be able to reach the entire load, so carefully loosen bag closures once they are in the autoclave. The dry goods without vacuum run simply pressurizes the chamber with steam for the duration of the cycle and then returns to normal. This process is primarily used for items that have been cleaned but need to be sterilized. Materials should be packed so that the heat and pressure can readily reach the whole load. Autoclaves generate extreme heat and high pressures, users should understand and respect the hazards these create. To prevent a sudden release of high-pressure steam, firmly lock autoclave doors and gaskets in place before you run the autoclave. Most, but not all autoclaves, have safety interlocks that prevent operation if the door isn't closed properly. Know if your autoclave has interlocks and take extra precautions if it is not equipped with interlocks. Some older autoclaves have little or no heat shielding around the outside. For these systems, attach Hot Surfaces, Keep Away warning signs to remind people of the hazard. Do not stack or store combustible materials (e.g., cardboard, plastic, volatile or flammable liquids, etc.) next to an autoclave. When operating an autoclave, follow these precautions: • Load the autoclave properly. Be sure to clean the drain strainer before loading. Don't load plastic materials that are not compatible with the autoclave. Individual glassware pieces should be within a heat resistant plastic tray on a shelf or rack, never place them directly on the autoclave bottom or floor. • Be sure the autoclave is OFF and the steam pressure is down before opening the door. • Open the door slowly, keeping head, face and hands away from the opening. • Wait at least 30 seconds after opening the door before reaching or looking into the autoclave. Before removing autoclaved items, wait 5 minutes for loads containing only dry glassware and 10 minutes for autoclaved liquid loads. • When removing items from the autoclave, wear heat-resistant, long-sleeved gloves and safety glasses or goggles treated with anti-fog solution. Remove solutions from the autoclave slowly and gently, some solutions can boil over when moved or when exposed to room temperature. • Let glassware cool for at least 15 minutes before touching it with ungloved hands. Be alert for autoclaved liquid bottles still bubbling. Let liquid loads stand in an out-of-the-way location for a full hour before touching them with ungloved hands. • Clean up any spills immediately.

26 Emergencies and Accidents

The University Public Safety Office responds to most routine emergencies on campus. If there is a life threatening emergency (e.g., fire, explosion, etc.), dial 9-911. This will connect you to the Vermillion 911 operator. Telephones on campus will also display the physical address of the caller. Dialing 911 from your cell phone will not provide the 911 operator with an address. In all emergencies and accidents, the first consideration is your safety and the safety of those around you. For non-emergency assistance on campus call USD Public Safety at 5342.

Preparation Be prepared for an emergency; know the hazards of all compounds/equipment you work with. Assess the risks before using any the chemical or biological compound. Where appropriate, post the proper procedures for handling the material and what procedures to follow in the event of a spill or other emergency. Emergency procedures should be posted in a conspicuous location to facilitate emergency responders’ assessment. For chemical hazards, the MSDS should be a primary reference for risk analysis. Risk analysis will include the following criteria: • Toxicity, reactivity and flammability of the compound both as an individual agent as in combination with other agents you may be using at the same time • The amounts involved. (Smaller is better) • The expected duration of your exposure to the compound. • Potential routes of entry for the chemical (i.e. inhalation, ingestion, injection, skin contact). Remember to include how your handling of the material may increase your risk of exposure. For example, weighing a powdered material often has a higher risk of exposure than pipetting a liquid form of the same chemical.

Make a Spill Kit Your lab should have stockpiled sufficient quantities of absorbents and other materials to control any spill that can be reasonably anticipated. Some uses such as hydrofluoric acid, mercury, biohazards, etc., require special equipment for emergency response. The general requirement is to protect yourself while you absorb / neutralize the spilled material and then clean the spill. Items needed in a spill kit include: • Personal protective equipment o 2 pair of chemical splash goggles o 2 pair of gloves (e.g., Silver shield, Nitrile, etc., a “universal” glove) o 2 pair of shoe covers o 2 plastic or Tyvek aprons and/or Tyvek suits • Absorbent materials o absorbent pillows / powders (e.g., 3M Powersorb or other commercial product) o activated carbon (good for organic solvents) o Floor-dry / Oil-dry (i.e., inexpensive absorbent similar to kitty litter) • Neutralizing materials o acid neutralizer

27 o caustic neutralizer (e.g., Neutrasorb [for acids] and Neutracit-2 [for bases], include a color change substance to indicate complete neutralization) o solvent neutralizer (e.g., Solusorb, activated carbon) to reduce vapors and raise the flash point of the mixture • Clean-up equipment o polypropylene scoop or dust pan o broom or brush with polypropylene bristles o 2 five (5) gallon polypropylene pails o 2 polypropylene bags o sealing tape o pH test papers o sign: Danger Chemical Spill – Keep Away

Chemical Spill: 1. Alert all persons nearby. 2. Assess the risk (i.e., small spill – one you can clean up - or big spill – one which requires outside assistance) a. If you know the material’s chemical properties and believe the size and hazard to be small, clean it up being sure to use appropriate PPE. Package and label absorbent / cleaning materials as hazardous waste. b. For major spill, one you cannot clean yourself, avoid breathing vapors and, for flammables, turn off ignition sources, if possible. Then, evacuate the area and close the door to the laboratory. Immediately notify your PI. c. If the spill occurs after normal hours and you cannot contact your PI, contact USD Public Safety (5342) for assistance.

Environmental Chemical Releases If a hazardous material spill gets out to the environment (floor drain, sink drain, ground, etc.), immediately contact your PI or USD Public Safety. Using absorbent material, attempt to stop or contain the spill/release without endangering yourself: 1. Extinguish all sources of ignition. 2. Isolate all potential environmental outlets (e.g. drains, sumps, soil, etc.). 3. Immediately notify your PI and USD Public Safety and wait for them to respond. It is important to record both the chemical compound spilled and the volume; this information will be needed for our report to the EPA.

Fire or Explosion: 1. Evacuate the fire area and close the door. 2. Notify occupants nearby. 3. Activate the building fire alarm system. 5. Evacuate and stay clear of building but close enough to provide information to emergency responders. Do not attempt to put out a fire unless you are experienced and trained in how to use the fire extinguisher.

Accidents and Injuries For serious injuries that require medical evacuation, immediately call 9-911 from any campus telephone. All other (minor) injuries should be assessed by a medical care provider (e.g., own

28 physician) and should be reported as soon as possible to the PI and the Dir. EHS or Dir. Human Resources. For potentially toxic chemical exposure, medical personnel should be provided with: • Identity of chemical(s) or biological - MSDS sheet • Conditions under which exposures occurred. • Signs and symptoms of exposure Exposure Monitoring and Medical Treatment

Regular environmental or employee exposure monitoring is not warranted or practical in laboratories because the chemicals used are often in well understood procedures, use is for relatively short periods of time and quantities generally small. Good laboratory procedures are carefully designed to ensure minimal exposure. Sampling may be necessary when highly toxic substances are used regularly or if it is mandated by regulation. Laboratory workers who suspect that they have been overexposed to a toxic chemical should report to their PI or the Director, EHS or call 9-911 (emergency) or 5342 (USD Public Safety non-emergency). Have a copy of the MSDS so that both signs and symptoms and medical treatment can be reviewed by competent medical personnel. A safety specialist will make an initial assessment and if needed, specific monitoring will be conducted.

Medical Examination and Consultation Medical examination and consultation is generally not needed for routine laboratory exposures. However, medical examination and/or consultation is recommended in the following situations: • A worker or visitor develops signs and symptoms of acute exposure. • An event takes place with the likelihood of an individual being exposed. • Required exposure monitoring results are higher than expected or allowed • There are special concerns about exposure to specific chemicals (e.g., reproductive toxins, carcinogens, etc.). • Personnel are engaged in laboratory animal handling and care or are handling human blood, body fluids or tissues. Training

Faculty members are responsible for insuring that their employees and students receive proper training. Handling, care and use of laboratory animals and radiation safety have specific training requirements. In addition to the training provided by faculty, the Howard Hughes Medical Institute has three training DVDs which are available as a training resource. • Practicing Safe Science o Overview program presenting safety instruction on chemical, physical, radiological and biological hazards in the molecular biology laboratory. Available online (practicesafescience.org) and DVD • Safety in the Research Laboratory o A nine-part series providing more detailed information providing guidance in recognizing and controlling hazards in a modern research laboratory. Available in video and DVD format. Program provides instruction in the following areas

29  Chemical hazards  Emergency Response  Radionuclide Hazards  Centrifugation Hazards  Chemical Storage Hazards  Glassware Washing Hazards  Mammalian Cell Culture Hazards  X-Ray Diffraction Hazards • Controlling Your Risks: HIV in the Research Laboratory o This program addresses the basic principles associated with biological safety with emphasis on HIV. Training requirements differ depending upon the actual hazardous material and work condition. In general, radiation, chemical and biosafety training should be given to new workers and a refresher class given annually. Records of training need to include the date, subject covered, and attendees. Each person receiving training should print and sign their name to acknowledge training. If a test is given to document competence, results of the test should be recorded or copies kept on file. As a minimum, safety training should include: • The chemical hygiene plan (e.g., this Biological & Chemical Safety Policy. • The location of MSDS and other hazard information in the laboratory. • The hazards of chemical and biological agents in the work area and how to protect against the hazards. • How to manage and dispose of waste or unwanted materials • How to detect the presence or accidental release of a hazardous chemical or biohazard and how to clean up a spill or disinfect a biohazard.

Training Records As noted above, training records associated with the USD Biological and Chemical Safety Program will be maintained by the PI and the person providing training, if different. These records will include the following information: • date of training session • contents or summary of the training • list of attendees • quizzes, if administered • names of persons conducting the training

30 Chemical Safety

Potentially hazardous chemicals can be found everywhere. There are an estimated 575,000 existing chemical products with hundreds of new ones introduced annually. Almost 32,000,000 workers are potentially exposed to one or more hazardous substance in the workplace. The fact that these same chemicals are available at your local hardware store does not mean they are without hazard. Many of these materials have properties that make them hazardous and are classed as physical (fire, explosion) and/or health (toxicity, chemical burns) hazards based on these properties. Depending upon magnitude, chemical exposure may cause or contribute to serious health effects including cancer, heart disease, burns, rashes, kidney and lung damage. There are many ways to work with chemicals which can both reduce the probability of an accident to a negligible level and reduce the consequences of an accident to minimum levels. Risk minimization depends on safe practices, engineering controls for chemical containment, the proper use of personnel protective equipment, the use of the least amount of a hazardous material necessary, and substitution of a less hazardous chemical for the more hazardous one. Understanding Chemical Hazards

To be classified as hazardous , a substance must be capable of producing adverse effects on humans or the environment. Before using any chemical, even if it is something that you have worked with at home or elsewhere, it is important to understand what the potential exposure hazards may be and how to use the chemical safely. To assess the hazards of a particular chemical, both the physical and health hazards of the chemical must be considered.

Physical Hazards of Chemicals The physical hazards of a chemical are those hazards inherent to a chemical's physical nature. Depending on its composition a chemical may be flammable, combustible, explosive, pyrophoric, oxidizer, compressed gas, cryogenic, etc. Flammability is the ability of a chemical to burn. Flammable and combustible chemicals evaporate rapidly and generate enough vapor to ignite in the presence of an ignition source (i.e., catch fire easily and burn readily). Flammable substances can be solid, liquid, or gaseous, but the most common type encountered in a laboratory is a flammable liquid or the vapor produced from such a liquid. Flammable chemicals are classified according to their flash point, boiling point and ignition temperature.
Flash point (FP ) is the lowest temperature at which a flammable liquid gives off sufficient vapor to ignite.
Boiling point (BP ) is the temperature at which the vapor pressure of a liquid is equal to the atmospheric pressure under which the liquid vaporizes. Flammable liquids with low boiling points generally present special fire hazards.
Ignition (or auto-ignition ) temperature is the lowest temperature at which a chemical will ignite and burn independently of its heat source. The lower the ignition temperature, the greater the fire potential. When the flammable vapor reaches its auto-ignition temperature, a spark is not needed for ignition. Additionally, flammable and combustible chemicals can react with oxidizers to cause a fire or explosion (i.e., a compound may burn so rapidly that it produces an explosion). Combustible materials will generate sufficient vapors at or above 38°C (100°F). Flammable chemicals will

31 generate sufficient vapors at temperatures below 38 °C (100°F). The table illustrates flammable and combustible class characteristics.

Flammable Combustible I-A I-B I-C II III > 23 oC (73 oF) > 38 oC (100 oF) > 60 oC (140 oF) Flash Point < 23 oC (73 oF) < 23 oC (73 oF) < 38 oC (100 oF) < 60 oC (140 oF) < 93 oC (200 oF) Boiling Point < 38 oC (100 oF) m 38 oC (100 oF) ------Flammable and combustible chemicals are also characterized by their explosive limits. The lower explosive limit (LEL ) or lower flammable limit is the lowest vapor concentration of the substance that will produce a flash of fire (i.e., blast) when an ignition source is present. The upper explosive limit (UEL ) or upper flammable limit is the highest vapor concentration of the substance that will produce a flash of fire when an ignition source is present. A substance's flammable range consists of all concentrations between the LEL and the UEL. The range may be dependent upon temperature and oxygen concentration. At higher concentrations than the UEL, the mixture is too rich to burn and at concentrations lower than the LEL, the mixture is too lean to burn. Generally, for flammable chemicals that are also toxic, concentrations at which the flammability is a hazard are usually well above the toxicity hazard concentrations. Oxidizers are chemicals which react with other substances, fuels or reducing agents, leaving them fewer electrons with which to maintain their atoms' required electron octets. The reaction may result in fire or explosion depending upon the nature of the fuel. Most oxidizers supply oxygen to common fires, but oxygen is not the only oxidizer supplied, fluorine and chlorine from oxidizers based on these electronegative elements will do as well. Chlorine based bleaches spilled on paper or wood may also combust. Common oxidizers found in labs include fluorine, chlorine, chlorite, borate, ozone, permanganate, nitric acid, chromic acid, hydrogen peroxide. Reactives are chemicals that readily react with ordinary unreactive chemicals such as air, water, cellulose, protein and steel, especially if the reaction is vigorous. Some chemicals, called self- reactive, even undergo change without any chemical input. Because reactive chemicals are likely to undergo vigorous, sometimes spontaneous, reactions and may spontaneously generate large quantities of heat, light, gases or toxic chemicals, work with reactive chemicals such as explosives, oxidizers, reducers, water reactives, and pyrophorics should be done only after understanding the possible reactions and potential energy release per mole. Pyrophoric chemicals can ignite on exposure to air at temperatures of 54 oC (130 oF) or lower. Such chemicals can be gases, solid or liquid, volatile or non-volatile. What occurs is that oxygen reacts with the chemical in the gas phase or on the surface in a way that leads to sustained combustion for as long as oxygen is present. Some examples are boranes, triethylaluminum, and white or yellow phosphorus. Many metallic powders are pyrophoric. The degree of reactivity is primarily related to particle size. Solids are more susceptible if finely divided or somewhat volatile. Compressed gases present chemical and physical hazards. If compressed gases are accidentally released, they may displace oxygen, create a fire or cause adverse health effects. Some gases (e.g., arsine, phosphine, phosgene, nitric oxide, chlorine, sulfur tetrafluoride, etc.), are potentially lethal if the cylinder leaks. If the leakage is of a flammable gas it can create an

32 explosive atmosphere. In a laboratory fire, the heat may cause the cylinder's internal pressure to increase and the cylinder may rupture. Cryogenic liquids are liquids with a boiling point that is less than or equal to -90 oC at one atmosphere pressure. Cryogenic fluids, such as liquid air, liquid nitrogen, or liquid oxygen, are used to obtain extremely cold temperatures. Most cryogenic liquids are odorless, colorless, and tasteless. Cryogenic liquids are hazardous because of the physical and chemical characteristics of their super-cooled state. Hydrogen, methane and acetylene gases are flammable, oxygen increases the flammability of combustibles, liquefied inert gases may displace oxygen in the air and all cryogenic liquids may produce extremely cold surfaces and may embrittle materials.

Health Hazards of Chemicals A chemical is called a health hazard if at least one study indicates that acute or chronic health effects may occur in exposed employees. Two terms are used when discussing the health effects of chemicals.
Toxicity is the ability of a chemical substance to produce injury once it reaches a susceptible site in or on the body. Toxicity is a property of each chemical.
Hazard is the probability that a substance will produce injury under the conditions / manner of use . The risk of injury is the probability that a chemical will cause harm. With proper handling, even highly toxic chemicals can be used safely. Conversely, less toxic chemicals can be extremely hazardous if handled improperly . Any substance can be harmful to living things. But, just as there are degrees of being harmful, there are also degrees of being safe. For every chemical, there are conditions in which it can cause harm and conditions in which it does not. The biological effects (beneficial, indifferent or toxic) of all chemicals are dependent on a number of factors: the route of exposure, rate, duration, frequency, total dose and the type of hazard. The most common route of entry for chemical substances is through inhalation (i.e., breathing). When breathed in, gases, vapors and particles can pass into the bloodstream along with oxygen or they may harm the tissues of the respiratory system (e.g., asbestos, silica, etc.). Chemicals that produce vapors should only be used in a well ventilated area or in a fume hood. Many chemicals have an odor which can be smelled at a certain concentration, called the odor threshold . However, olfactory fatigue may occur when a worker has been exposed to high concentrations or after prolonged lower level exposure and may make an odor seem to diminish or disappear, while the danger of overexposure still remains. Overexposure symptoms may include headache, increased mucus production, and eye, nose and throat irritation. Narcotic effects, such as confusion, dizziness, drowsiness, or collapse, may result from exposure to some substances, including many common hydrocarbon solvents (e.g., toluene). In the event of overexposure, close containers, open windows or otherwise increase ventilation, and move to fresh air. If symptoms persist, seek medical attention.

33 The second most common route of entry is skin or eye absorption of chemical solids, liquids, vapors, and gases. Skin contact may produce a local reaction (e.g., burn or rash) but can result in absorption into the bloodstream with no skin reaction. Absorption into the blood may allow the chemical to cause toxic effects on other parts of the body. The absorption through intact skin is influenced by the health of the skin and the properties of the chemical. Skin that is dry or cracked or has small cuts or lacerations offers less resistance. Wear gloves and other protective clothing to minimize skin exposure. Symptoms of skin exposure may include dry, whitened skin, redness and swelling, rashes or blisters, and itching. In the event of chemical contact on skin, rinse the affected area with water for at least 15 minutes, removing contaminated clothing while rinsing. Seek medical attention if symptoms persist. Chemical contact with eyes can be particularly dangerous and may produce a painful injury or even blindness. Wearing safety goggles or a face shield can reduce the risk of eye contact. Eyes which have been in contact with chemicals should be immediately rinsed with water continuously for at least 15 minutes The third common route of entry into the body is ingestion (i.e., swallowing). Ingestion can occur by failing to wash hands before eating or drinking, eating or drinking contaminated food or beverages in the work area, or touching the mouth with contaminated hands. Workers can easily reduce the risk of ingestion by not eating, drinking nor storing food in the areas where chemicals are used or stored. Additionally, washing hands thoroughly after working with chemicals, even when gloves are worn, reduces the risk of cross contamination. A fourth route of exposure is by accidental injection which can occur by needle sticks or through accidents with broken glassware or other sharp objects that have been contaminated with chemicals. If accidental injection has occurred, wash the area with soap and water and seek medical attention, if necessary. To reduce this risk, always use caution when handling sharp objects.

Toxic Exposure Factors While the daily use of many chemicals can be perfectly safe, the body normally reacts to exposure from harmful chemicals. Toxic effects of chemicals can range from mild and reversible (e.g., a headache from a single episode of inhaling the vapors of petroleum naphtha that disappears when the victim gets fresh air) to serious and irreversible (liver or kidney damage from excessive exposures to chlorinated solvents). A goal of chemical safety is to make workers aware of health hazards of the chemicals they are using. The toxic effects of a chemical may be local or systemic. • Local injuries only involve the area of the body in contact with the chemical. For example, if you spill an acid on your arm, the effect will be on your arm. • Systemic injuries involve tissues or organs away from the contact site where the toxic substance has been transported through the bloodstream. For example, methanol that has been swallowed may cause blindness. Certain chemicals may only affect a target organ . Lead primarily affects the brain, kidney and red blood cells while some organic solvents may harm the liver and kidneys. It is also important to distinguish between acute and chronic exposure / toxicity.
Acute toxicity results from a single, short intense exposure to a chemical where the acute effects usually appear quickly and may be reversible. Hydrogen cyanide, hydrogen sulfide, nitrogen dioxide, ricin, organophosphate pesticides and arsenic are examples of acute

34 toxins. Do not work alone when handling acute toxins. Use a fume hood to ensure proper ventilation.
Chronic toxicity results from repeated exposure to lower levels over a long period of time. The effects are usually delayed and gradual, and may even be irreversible. Mercury, lead, and formaldehyde are examples of chronic toxins. People react differently in their sensitivity to chemical exposure. This individual susceptibility to chemicals depends on factors such as: age, sex, eating habits, physical condition, medical conditions, drinking and smoking, pregnancy, etc. Over time, regular exposure to some substances may lead to the development of an allergic rash, breathing difficulty, or other reactions. This physical response is referred to as sensitization . Continuing exposure past this point and the effects may occur with exposure to smaller and smaller amounts of the chemical. With sensitization, the effects usually disappear soon after the exposure stops. For reasons not fully understood, not everyone exposed to a sensitizer will experience this reaction. Examples of sensitizers include epoxy resins, nickel salts, isocyanates and formaldehyde. Some workers even become sensitized to the rubber used in protective gloves where their hands may begin to itch. Some workers have even experienced life- threatening shock reaction from gloves made from natural latex. The physical class of the substance (i.e., solubility ) is also a key exposure factor. Highly soluble materials like ammonia irritate the upper respiratory tract while relatively insoluble materials like nitrogen dioxide penetrate deep into the lung. Fat soluble materials like pesticides tend to have longer residence times in the body. An aerosol is composed of solid or liquid particles of microscopic size dispersed in a gaseous medium. The toxic potential of an aerosol is only partly described by its concentration (mg/m 3). It is also necessary to know the particles size. Particles above 1 micrometer (1 mm) tend to deposit in the upper respiratory tract. Particles less than 1 mm in diameter enter the lung. Very small particles (< 0.2 mm) are generally not deposited.

Types of Health Hazards Toxic chemicals used in labs can produce physical effects. The physiological classification of toxic materials include: allergens and sensitizers, irritants, corrosives, asphyxiants, anesthetics, hepatotoxic agents, nephrotoxic agents, neurotoxic agents, agents which affect the hematopoietic system, fibrosis-producing dusts, carcinogens, mutagens, or teratogens. Many chemicals have multiple toxic and/or hazardous characteristics. Prior to working with a chemical you need to determine the answers to questions like: Is the chemical toxic? How toxic is it? Are you exposed to it? Does that exposure represent a risk to your health? What kind of risk? Find out all the information that you can about the health risks of the chemicals that you

35 work with. Read the Material Safety Data Sheets (MSDS) and review the characteristics of the chemicals you plan to use.

Reproductive Health Hazards Some chemicals can cause damage to the reproductive systems of men or women leading to infertility, impotence, menstrual irregularities, spontaneous abortion or damage to offspring. Reproductive health hazards include heavy metals, some aromatic solvents (benzene, toluene, xylenes, etc.), and some therapeutic drugs. Reproductive toxins are substances that cause chromosomal damage ( mutagens ) and substances with lethal or teratogenic (malformation) effects on fetuses. Embryotoxins or fetotoxins are substances that may be lethal to the fertilized egg, embryo or fetus, may be teratogenic (i.e., cause fetal malformations), may retard growth or may cause postnatal functional deficits. Male reproductive toxins can in some cases lead to sterility. Two well known male reproductive toxins are ethylene dibromide and dibromochloropropane. When a pregnant woman is exposed to a chemical, some of the chemical may cross the placental barrier affecting the fetus. A fetus may be more sensitive to some chemicals than the mother; especially during the first twelve weeks of pregnancy when the mother may be unaware she is pregnant. Teratogenic chemicals are those substances that cause fetal death or malformation from maternal exposure during pregnancy. These teratogens are agents which interfere with normal embryonic and fetal development without apparent damage to the mother or lethal effects on the fetus. Because cellular genetic effects are not produced, these effects are not hereditary. Known human teratogens include organic mercury compounds, lead compounds, 1,2-dibromo- 3-chloropropane, ionizing radiation, some drugs, alcohol ingestion, and cigarette smoking. Some substances which may cause adverse reproductive effects in males include 1,2-dibromo-3- chloropropane, cadmium, mercury, boron, lead, some pesticides, and some drugs. More than 800 chemicals have been shown to be teratogenic in animal models; many of these are suspected human teratogens. Embryotoxins are substances that act during pregnancy to cause adverse effects on the fetus. These effects include death of the fertilized egg, the embryo, or the fetus, malformation, retarded growth and postnatal functional deficits. Examples of embryotoxins include organomercurials, lead compounds, and formamide. A mutagen affects the genetic material of exposed cells. Mutations can occur on the gene level (gene mutations) as when one nucleotide base-pair is change to another. Mutations can also occur on the chromosomal level (chromosomal mutations) when the number of chromosomal units or their morphological structure is altered. The effect is inherited by daughter cells and, if it occurs in the gonads or reproductive organs, can become part of the genetic pool that is passed on to future generations. Examples of mutagens commonly found in research laboratories include: ethidium bromide, barium permanganate, methyl isocyanate and radioisotopes.

Particularly Hazardous Substances A group of chemicals have been evaluated and determined to have the potential to be particularly hazardous. These include acutely toxic substances, selected carcinogens and reproductive toxins. Labs with these substances must take the additional precautions when working with these agents to protect workers.

36 Toxicity Relationships All chemicals are toxic under some condition of exposure. In order to compare the toxicity characteristics of chemicals, it is necessary to define these exposure conditions as well as the quantity involved in the exposure. Acute toxicity is the ability of a chemical to cause a harmful effect after a single exposure to the substance by any route for a short (i.e., acute) period of time (e.g., less than one day). Acute toxicity information consists of (1) lethality data, the levels of exposure (LC 50 ) or dose (LD 50 ) estimated to kill 50 percent of a specific population of animals under controlled conditions and (2) dose-response (mortality) relationships. On MSDSs, the degree of acute toxicity is expressed by these acronyms:

LD LO The lowest dose of a material introduced by any route, other than inhalation, over any given period of time in one or more divided portions and reported to have caused death in humans or animals.

LC LO The lowest concentration of a material in air, which has been reported to have caused death in humans or animals.

LD 50 Lethal Dose to 50 percent of a population (of lab animals). The amount of dose, in mg/kg of body weight, to kill one-half of the animals to which it is administered. This is widely

used as an index of toxicity. The lower the LD 50 , the more toxic the substance. LC 50 Lethal Concentration to 50 percent of a population (of lab animals). Refers to an airborne concentration of a contaminant that will kill one-half of the population of study

organisms. Used as an index of toxicity. The lower the LC 50 , the more toxic the substance. Categories of acute lethal toxicity have been developed by toxicologists. An example of such a classification is given in the table. Classes of Acute Toxicity Dose (amount of Rat Oral

Toxicity substance per kg Probable Oral Lethal Dose LD 50 of Class of body weight) (for a 70 kg Adult Human) Examples Example Practically > 15 g/kg More than 1 quart Sucrose 29.7 g/kg nontoxic Slightly 5 - 15 g/kg Between a pint and a quart Ethanol 14 g/kg toxic Moderately Sodium 0.5 - 5 g/kg Between an ounce and a pint 3 g/kg toxic Chloride Very Between a teaspoonful and an 50 - 500 mg/kg Caffeine 192 mg/kg toxic ounce Extremely Between 7 drops and a Sodium 5 - 50 mg/kg 6.4 mg/kg toxic teaspoon Cyanide Supertoxic < 5 mg/kg A taste (less than 7 drops) Strychnine 2.5 mg/kg If any of the following criteria are satisfied for a particular chemical, then it is considered very toxic :

A chemical that has a median lethal dose (LD 50 ) of more than 50 mg per kilogram, but not more than 500 mg per kilogram of body weight when administered orally to rats.

A chemical that has a median lethal dose (LD 50 ) of more than 200 mg per kilogram but not more than 1000 mg per kilogram of body weight when administered by continuous contact for 24 hours (or less if death occurs within 24 hours) with the bare skin of rabbits.

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A chemical that has a median lethal concentration (LC 50 ) in air of more than 200 ppm but not more than 2000 ppm by volume or less of gas or vapor, or more than 2 mg per liter but not more than 20 mg per liter of mist, fume, or dust, when administered by continuous inhalation for one hour (or less if death occurs within one hour) to rats.
Additionally, if any of the following criteria are satisfied for a particular chemical, then it is considered extremely toxic:

A chemical that has a median lethal dose (LD 50 ) of 50 mg or less per kilogram of body weight when administered orally to rats.

A chemical that has a median lethal dose (LD 50 ) of 200 mg or less per kilogram of body weight when administered by continuous contact for 24 hours (or less if death occurs within 24 hours) with the bare skin of rabbits.

A chemical that has a median lethal concentration (LC 50 ) in air of 200 ppm by volume or less of gas or vapor, or 2 mg per liter or less of mist, fume, or dust, when administered by continuous inhalation for one hour (or less if death occurs within one hour) to rats. Chronic toxicity is the toxic effect resulting from repeated, low-level daily doses over a person's or animal's lifetime. These chronic effects can result from cumulative damage to tissues sustained from each small dose, or they can result from accumulation of the toxic chemical in the body over a long period of low-level exposure (e.g., mercury, lead). Latent effects, such as carcinogenicity or mutagenicity, are examples of long term or chronic effects. The damage done from one large or multiple low-level exposures to a carcinogen is often delayed. Thus, a cancer may not show up until after a 10 to 20 year latent period has elapsed. Likewise, the effect of exposure to a mutagen may not manifest itself until the birth of offspring with malformations resulting from the mutation. Material Safety Data Sheets (MSDS) and Labels

OSHA requires chemical manufacturers and suppliers provide users with Material Safety Data Sheets (MSDS ). These are designed to provide the information needed to protect workers from hazards associated with the chemical. Labs are required to have MSDSs for all chemicals and make them readily available to workers. The MSDS provides a variety of fundamental health and safety information related to the chemical. This information will allow the user of the chemical to recognize and prepare for potential hazards associated with the chemical and prepare for and react to emergency situations. Because of all the information they contain, workers should then review the MSDS for each of the unfamiliar chemical they will use. Although OSHA does not mandate a specific format for the MSDS, manufacturers normally publish their MSDS using either the OSHA Form 174 or the American National Standards Institute (ANSI) suggested format. Regardless of the format used, the information that will be found on an MSDS includes:

• Name of supplier (with address and phone number) and date MSDS was prepared or revised -- because toxicity data and exposure limits may be revised, the MSDS should be reviewed

38 to insure information is still correct. The phone number provides a point-of-contact for additional information on hazards and emergency response. • Name of the chemical -- The identity of the substance as it appears on the label, for mixtures, this may include the identity of most, but not every ingredient. Common synonyms are often listed. • Physical and chemical properties -- boiling point, molecular weight, vapor pressure, vapor density, specific gravity, melting point, evaporation rate, solubility in water, physical appearance and odor. • Physical hazards - related to flammability, reactivity, and explosibility. Flammability information may include flash point (and method used to determine it), flammability limits, extinguishing media, special fire fighting procedures, unusual fire and explosion hazards. Reactivity includes stability, conditions to avoid, incompatibility (materials to avoid), hazardous decomposition or byproducts, hazardous polymerization (and conditions to avoid). • Toxicity data -- OSHA and American Conference of Governmental Industrial Hygienist (ACGIH) exposure limits are listed. Lists the hazardous components by chemical identity and other common names. Includes OSHA Permissible Exposure Limit (PEL), ACGIH Threshold Limit Value (TLV) and other recommended exposure limits. May also include the percentage listings of the hazardous components. • Health hazards -- Acute (immediate) and chronic (build up over time) health hazards, routes of entry (inhalation, skin, ingestion), carcinogenicity (NTP, IARC monographs, OSHA regulated), signs and symptoms of exposure, medical conditions generally aggravated by exposure, emergency and first aid procedures. • Storage and handling procedures -- precautions for handling or storage and listing appropriate control measures including respiratory protection, ventilation (local, mechanical exhaust, special or other), protective gloves, eye protection, other protective clothing or equipment, work/hygienic practices, first aid treatment, steps to take if the material is released or spilled, and guidelines for the proper disposal of waste material. Because the MSDS is written to address the widest conceivable use of the material, the recommended procedures may be more stringent than necessary for laboratory use. Labeling Systems Labels on containers is another source of safety information. There are several standardized labeling systems which workers may see in the work place. The most commonly types of labeling system encountered are the Department of Transportation (DOT), the National Fire Protection Association (NFPA), and the Hazardous Materials Information Systems (HMIS). DOT Labels - When chemicals are shipped or transported in the public sector, there must be labels to satisfy DOT rules. Many larger containers (e.g., 5-gallon cans and 55-gallon drums) are also individually labeled with the standard DOT labels. These labels are designed to identify DOT classes of hazardous materials. These DOT labels are diamond- shaped and color-coded by hazard. The hazard class or division number appears in the lower corner. The primary purpose of the DOT label is to provide a warning to first responders at a transportation mishap about the chemical hazard the material poses. NFPA Labels - The popular NFPA diamond was developed by the National Fire Protection Association to aid emergency responders in recognizing potentially hazardous situations. The label contains 4

39 colored diamond shapes. Each colored diamond is associated with a different type of physical or health hazard. However, because this system refers to the hazards associated with the material under fire-type conditions, the information is of limited value for routine laboratory use of the chemical. HMIS Labels - Another popular system was developed by the National Paint and Coatings Association. It contains 4 different colored rectangular shapes that are related to different hazards. As opposed to the NFPA label, the Hazard Materials Information System (HMIS) rates the material risks under normal use conditions. Comparison of Labels Workers will probably see both types of labels used on containers. At first glance, these labeling systems appear quite similar. On both the NFPA and HMIS label, each color represents a specific type of hazard: − Blue stands for health hazard − Red means flammability hazard − Yellow is reactivity hazard (NFPA) or Orange is physical hazard (HMIS) − White stands for special hazard information or special notice The blue, red, and yellow/orange sections also contain a number from 0 to 4 that tells the degree of hazard. The number 4 is for the most serious hazard, 0 the least serious. The white section of the label uses no numbers. If a material presents a special hazard, then a symbol or phrase may be placed in the white section giving special attention. The HMIS label will sometimes note personal protective equipment using specially designated icons. However, there are significant differences between the two systems. The HMIS system attempts to convey full health warning information to all employees while the NFPA diamond conveys hazard information to fire fighters and other emergency responders. HMIS is not intended for emergency circumstances. The current version of the HMIS, called HMIS III replaced an earlier system. The revision replaced a yellow Reactivity section (similar to the NFPA reactivity section) with the orange Physical Hazard section. While both types of labels may be seen, the label with the yellow Reactivity type label will ultimately vanish. The HMIS label attempts to convey full health warning information to the user just as it is listed on an MSDS. The information each of the sections of the label provide.

40 Health - This conveys the health hazards of the material. The blue Health bar has two spaces, one for an asterisk and one for a numeric hazard rating. The asterisk, if present, indicates the substance is a chronic health hazard , meaning that long-term exposure to the material could cause a health problem such as emphysema or kidney damage. The NFPA diamond lacks this "chronic" information because NFPA is meant only for emergency or acute (short-term) exposures. The numbering system uses a 0 to 4 scale where 0 indicates minimal hazard and 4 indicates an extreme hazard. Flammability - Initially the NFPA and HMIS used the same criteria to assign numeric values (0 = low hazard to 4 = high hazard). In HMIS III, the flammability criteria are defined according to OSHA standards which defines a flammable liquid as "any liquid having a flash point below 100 oF (37.8 oC), except any mixture having components with flash points of 100 oF (37.8 oC) or higher, the total of which make up 99 percent or more of the total volume of the mixture. Flammable liquids shall be known as Class I liquids." Physical Hazard - HMIS III replaced the Reactivity (yellow) rating of HMIS II with an orange section using the OSHA criterion of physical hazard. Seven such hazard classes are recognized: − water reactives − compressed gases − oxidizers − organic peroxides − pyrophoric materials − unstable reactives − explosives The numerical rating values of 0 = low hazard / stable to 4 = high hazard / may detonate, are used to describe the magnitude of hazard from the substance. Personal Protection - This is by far the largest area of difference between the NFPA and HMIS systems. The white diamond in the NFPA system is used to convey special hazards (e.g., water reactive, oxidizer, radioactive, etc.). The white section in the HMIS label indicates the personal protective equipment that should be worn when working with the material. The HMIS Personal Protection coding system uses a letter coding system to prescribe the required protective equipment. The reason this is a drawback is that some of the letters / symbols used are used by other hazard communication systems and have completely different meanings and applications. For example, consider the NFPA labeling for a container of methanol. A hazard degree rating of 1 has been assigned to the health hazard which means the material is a slight hazard. The flammability rating is 3, meaning it has a flash point below 100 oF and will readily ignite at normal temperatures. The reactivity rating of 0 means that the material is stable. Personal protective equipment includes safety goggles, rubber gloves and apron. Other symbols which might be used include no smoking, no open flames, no matches. Since it is poisonous, the "skull & crossbones" symbol or the word "poison" would appear on the NFPA label. The target organs affected from a hazardous exposure are blood, eyes, intestines, and stomach. Other Labeling Systems - While the NFPA and HMIS systems are relatively common, some vendors have created their own label system which incorporates information from one or both

41 of these labels. When looking at containers, look for the common elements rather than the differences (e.g., are the hazard ratings high or low, special equipment, etc.). Labeling Secondary Containers Sometimes workers need only a small amount of material for a specific task and may transfer the amount of chemical they need from the original container to a smaller, more portable, secondary container . If all of the material is to be used immediately by the employee who transferred the material, the secondary container need not have a label. However, the chemical can only be used by the worker who transferred it and it must be only used on that shift. It is better to label any secondary container with all the necessary information. Laboratory Safety Procedures

Step back and look at your laboratory as if you were seeing it for the first time. This is the view that new faculty, staff, students and visitors to your lab see. Does it look safe? Is it neat and orderly? Are people taking safety precautions? Is there a strong odor of volatile chemicals? Does it look like chemicals are stored safely? Can you see ways to make your lab safer? Better yet, conduct a more systematic survey of your laboratory's safety practices by using one of the survey forms in Appendix E. Previously we discussed chemical physical and health hazards. When you plan laboratory procedures, use that guide and these basic principles to help assure safety:
Know about the chemicals and hazards associated with your lab. Know their potential flammability, reactivity, corrosivity and toxicity. Know how to read and interpret MSDSs.
Know what to do in various emergency situations (e.g., fire, chemical spill, injury, etc.).
Avoid working alone in a laboratory.
Don't underestimate risks; assume any mixture will be more hazardous than its most hazardous component and that all substances of unknown toxicity are toxic.
Minimize all chemical exposures. Few laboratory chemicals are without hazards. Use precautions when handling all laboratory chemicals. Wear personal protective equipment appropriate to the work. Do not wear contact lenses around chemicals, fumes, dust particles or other hazardous materials.
Use extreme care when working with needles, blades and glass.
Do not eat, drink, apply makeup or use tobacco products in the laboratory. Do not mouth pipet. Do not use ice from a laboratory ice machine for human consumption. Dedicate microwave ovens and other heating devices exclusively for food or for laboratory operations. Ensure that ovens are clearly labeled to indicate their function.
Provide adequate ventilation. The best way to prevent exposure to airborne substances is to vent them away from you. This is accomplished by using fume hoods and other ventilation devices. Avoid using dry ice in enclosed areas; it can produce elevated carbon dioxide levels. Dry ice mixed with isopropanol or ethanol may cause frostbite. Avoid producing aerosols.
Protect unattended operations from utility failures and other potential problems that could lead to overheating or other hazardous events.
Clean contaminated equipment and spills immediately. Avoid contaminating equipment with mercury and clean mercury spills immediately. Use non-mercury thermometers when appropriate.

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Keep hallways, corridors and exit ways clear. Do not locate laboratory equipment or supplies in these areas.

Laboratory Safety Surveys How do you know if your laboratory is a safe place? One way to find out is to audit your laboratory for safety practices and facilities. This can be easily accomplished by using the Laboratory Safety Survey in Appendix D. Other audit / survey resources are available. The state Risk Management Office inspects the campus every few years. The University Public Safety Office conducts annual life safety audits of all spaces and monthly inspects portable fire extinguishers. As required by the fire code, fire alarms are tested and fire suppression systems are inspected semiannually. These audits and tests help us prevent fires by looking for problems, like improper or excess storage of flammable liquids. Environmental Health & Safety (EHS) is always available to help you with safety problems and it annually performs safety surveys of laboratories. Upon request, EHS can also review a specific area or practice or perform an overall survey of your building. The annual audit involves: • Discussing your concerns and safety issues. • Touring your areas to assess the risks to health, property or the environment. • Comparing your practices and facilities to current and accepted safety standards. • Discussing solutions, including employee training, personal protective equipment, improved ventilation or further testing and evaluation.

Good Housekeeping Facilitates Safety Generally speaking, a clean, orderly lab is a safe lab. Good housekeeping can lower the number of lab accidents and reduce the risk and consequences of a fire. It can also increase your working space. For a safe and efficient laboratory:
Keep passageways to exits clear.
Do not block areas around safety showers, fire extinguishers, fire blankets and electrical (on/off) controls.
Do not store chemical containers on the floor where they may be broken or become a trip hazard for workers.
Return chemical containers to their proper storage location after use.
Do not use floors, stairways or hallways as storage areas.
Do not store flammable materials and empty boxes up to the ceiling. Dispose of empty boxes, you can always find one or two if needed. Otherwise, fold boxes flat and store.
Keep balances, hoods, centrifuges, incubators, refrigerators, ovens and other common-use items clean and neat for the next user.

Laboratory and Personal Hygiene Good personal hygiene will help minimize exposure to hazard chemicals. Laboratory hygiene consists of practices to avoid accidental or inadvertent exposure to laboratory chemicals. If there is a small spill of a fine powder, a careless coworker can quickly spread contamination throughout a laboratory. Even small exposures from some compounds may result in harmful effects for people who work in laboratories every day. Your goal should be no skin contact with laboratory chemicals. Unlike radioactive materials, chemical contamination is difficult to detect. When fluorescent dyes have been spilled in University buildings, it is unnerving to see how

43 widely the contamination spreads. To keep yourself and others safe from accidental contamination: • Do not touch things that are used by non-gloved hands (e.g., telephone, door knobs, etc.) if you are wearing gloves that have touched chemicals. Gloves can be washed with soap and water in many cases if removal is not convenient. • Wash your hands in the lab frequently, especially after lab work and before eating, drinking, applying makeup or leaving the area. • Routinely wash doorknobs, telephones, keyboards and desks. • Never use mouth suction to fill a pipet. Use a pipet bulb or other mechanical pipet filling device. • Don't keep food in refrigerators used to store chemical, radioactive or biological experiments. Do not use laboratory equipment to serve or store food or drinks. • Never eat or drink in labs. • Do not wear contact lenses near chemicals, especially corrosives or volatile solvents. This used to be a prohibition but the recommendation has been relaxed, theoretically safety glasses will be safer than contact lenses. • Never allow a laboratory chemical to touch your skin; use gloves and wear a lab coat. Remove contaminated clothing immediately and do not use the clothing again until it has been properly decontaminated. • Don't immerse your fingers or hand in liquids; use tongs or a tool. • Do not sniff or taste chemicals.

Chemical Storage and Management Proper chemical storage is as important to safety as proper chemical handling. Don't just store your chemicals; your stocks and inventory of laboratory chemicals need to be actively managed. From the time laboratory chemicals are received in your lab to the time of disposal, inventory them, separate incompatibles, store them safely and regularly review their status.

Chemical Inventory Your lab is in many ways like your home. Many people shop at the grocery store using a shopping list of food stuffs and consumables to buy. This shopping list is filled out by taking inventory of what you have on hand and what your plans are for the immediate future. In shopping from this list you consider things like, "When does the milk expire?" "If I buy 4 of these, how long will they last?" Prudent management of chemicals in your lab is facilitated by an inventory. Such an inventory saves money because a knowledge of how much of what chemicals are on hand prevents the purchase of duplicates. If you are aware of usage levels, materials can be routinely ordered. An inventory can help you monitor chemicals that degrade with age (e.g., ethers, gas cylinders, etc.), and can help you keep incompatibles separate and to anticipate special storage requirements that certain chemicals may have. Additionally, a summary of chemical types may provide emergency responders with a measure of hazards to be encountered within that lab. You can keep information on each chemical container using file cards or a computer database / spreadsheet. A computer database can be shared with other laboratories in the department / school to advertise surplus stocks and prevent redundant purchasing for the entire department.

44 Keep track of your chemical purchases by saving invoices. Monitor chemical use by keeping track of empty bottles. Keep track of chemical disposal by retaining copies of surplus chemical and carboy forms. For your safety, it is important that you know your chemicals and the hazards they pose to you. For each chemical you purchase, you should also receive a Material Safety Data Sheet (MSDS). If you don't receive an MSDS, call the supplier or the EHS for a copy. Additionally, the Safety Department web page (http://www.fpm.wisc.edu/safety) has links to MSDS sites. Keep a binder of your MSDS pages for everyone in your lab to use. Upon receipt of new compounds, review the MSDS to determine the hazards of the chemical. Chapter 2 of this Guide can help you interpret the MSDS and other hazard information. Make sure the container is labeled with the chemical's hazard, so that others know too. On receipt is also a good time to update the inventory. In a large lab, the most effective system is to have a computerized inventory with each record corresponding to a single container. Essential inventory information includes:
the chemical name as printed on the container,
molecular formula (for further identification and to simplify searching),
Chemical Abstract Service (CAS) registry number (unambiguously identifies chemicals regardless of different names),
lot number
source, and
size of container. Other useful information for the spreadsheet database is: • hazard classification to guide in safe storage, handling and disposal, • date of acquisition to insure unstable compounds are not stored beyond their useful life and to insure older chemicals are used first, • storage location (if multiple locations exist).

Chemical Storage Principles Storing chemicals requires the integration of several basic health and safety considerations. The inventory system goes a long way to assure safe storage along the lines of physical safety and chemical compatibility. Follow a few other basic considerations to assure safety. Read chemical labels and MSDSs for specific storage instructions. The supplier of your chemical has already investigated safety issues and listed these on the MSDS and label. Store your chemicals in a safe place. Store chemicals in a well-ventilated area; however, don't store chemicals in a fume hood . Keep your fume hood clear for work requiring a fume hood. Use a ventilated cabinet to store volatile and odorous chemicals. Use sturdy shelving with ample space for every container. Never store hazardous chemicals in a public area or corridor. Don't store liquids above solids. To avoid the risks of lifting and reaching, keep large and

45 heavy items on lower shelves. Store glass containers so that they are unlikely to be broken. Keep containers off the floor, safe from an accidental kick. Use plastic trays for secondary containment to contain liquid spills. Think about ways to keep your chemicals from spilling and contained if they do spill. Avoid storing chemicals on shelves more than six feet above floor. Store all hazardous chemicals below eye level so you can easily read the label. Do not store liquids above eye level. Fire codes require that nothing be stored within 18 inches of a fire sprinkler head on the ceiling. Keep incompatible chemicals separate. Group chemicals according to their hazard category (i.e., acids, bases, flammables, etc.) to prevent chemical reactions and insure compatible storage. A suggested shelf storage pattern published in the NIOSH School Chemistry Laboratory Safety Guide (2006) is at Appendix B. Some general guidelines include: • Separate acids from bases, store these chemicals near floor level • Isolate perchloric acid from organic materials. Do not store perchloric acid on a wooden shelf. • Separate highly toxic chemicals and carcinogens from all other chemicals. Place a warning label on this storage location and keep it locked. • Separate acids from flammables. • Do not keep peroxide-forming chemicals longer than 12 months. • Do not allow picric acid to dry out. • If flammables need to be chilled, store them in a laboratory safe refrigerator, not in a standard refrigerator. • Flammables should be stored in a flammable storage cabinet. Label all chemical containers. If you make solutions, synthesize products or transfer chemicals to another container, make sure all containers are labeled. Clearly label each chemical container in your laboratory with: − the chemical name − the principal hazard (e.g., carcinogen, irritant, corrosive, etc.) − the date prepared, opened or received − initials of the person making the label OSHA and EPA rules and regulations imply that if a container has an expiration date on the label, then the chemical must be used or disposed by that date. If you are going to hold chemicals past their expiration date, the viability of the chemical compound should be evaluated and documented for containers past their "expiration" date (e.g., write on the container a reevaluated expiration date and initial it). Unfortunately, there are some routine laboratory environmental conditions that contribute to deface or obliterate labels: • hydrogen chloride vapor from neighboring chemicals. • Iodine from chemicals that hydrolyze to iodide and that is oxidized in air. • light, fluorescent and sun light, acts on red print. • embrittlement of paper. • cellophane tape over paper, tape discolors easily.

46 • glue oxidizes. • mold grows on labels of bottles in cold rooms that condense air humidity. While all chemical containers must be labeled to prevent the hazards and disposal problems associated with unknown chemicals, labeling of many small vials with complete chemical names can be a difficult and tedious task. To make this job easier, use these tips.
Label the entire group. If you have a rack with vials that hold various fractions from a column, label the entire rack with a description of what is contained in the individual vials.
Refer to your notebook. Give the containers numbers that are referenced in your laboratory notebook. Inspect storage areas periodically. When a lab is being cleaned out, it is not unusual to find chemical containers that are more than 20 years old, some with labels that are unreadable. Storage areas should be inspected at least annually. Remove unwanted or expired chemicals and update the inventory. Visually inspect stored chemicals to determine viability and safety. Chemicals showing any of these indications should be evaluated for disposal: • slightly cloudy liquids • pressure buildup in containers • darkening or change in color • evidence of reaction with water • spotting on solids • corrosion / damage to container • caking of anhydrous materials • missing / damaged / illegible labels • existence of solids in liquids or liquids in solids

Storage of Flammable and Combustible Chemicals Suppose you came home one day and found two 5-gallon cans of gasoline and several jars of paint thinner in your kitchen. Your first concern would be to move the containers somewhere outside the house, making sure that you didn't break any. Then open the doors to remove the smell. Why would you be less concerned with such quantities of highly flammable liquids in your lab? Chemical labels and Material Safety Data Sheets will help you identify flammable and combustible chemicals. Improperly stored flammable and combustible chemicals can provide the fuel that can lead to a catastrophic laboratory fire. Use the following guidelines for storing flammable chemicals: • Minimize the amount of flammable liquids in your lab. Buy only what you will use in the immediate future, and buy the smallest size that you need. Excess flammable solvents risk a fire, a dangerous spill and, if you are exposed to them, your health. Unused surpluses may cost the University thousands of dollars for disposal. • If a building or departmental flammable solvent storage room with a fire suppression system is available, store flammable materials there until you need to use them and remove only the amount needed for a particular experiment or task.

47 • In the laboratory, store flammables in a UL-approved (or equivalent) flammable storage cabinet. Unless a cabinet is marked as approved for storage of flammable liquids, flammable solvents may not be stored there. In general, do not store flammable liquids in cabinets below fume hoods or sinks. • Store flammables, combustibles and other fuels away from strong oxidizers, such as perchloric and nitric acids. It is best to store flammable liquids in an approved storage cabinet dedicated solely for that purpose. • Limit quantities of flammable liquids stored outside of safety cans and flammable storage cabinets to less than ten gallons per one hundred square feet (i.e., per lab suite). If you include flammables stored in safety cans and flammable storage cabinets, limit the amount of flammable liquids to less than twenty gallons per one hundred square feet of lab space. Thus, the maximum quantity of flammable liquids in each lab suite / fire area depends upon the storage configuration: − Glass, metal or plastic 10 gallons (38 liters) − Safety cans 25 gallons (95 liters) − Flammable liquid storage cabinets 180 gallons (684 liters) • On your benchtop, limit the storage of flammable liquids to those in immediate use. Handle flammable chemicals in areas free from ignition sources. • It is best to store bottles of (flammable) liquids in a tray or pan (secondary containment) to catch any spills. • Use plastic trays when storing chemicals in freezers. This prevents the bottles from becoming embedded in ice and frost that often forms in freezers. It also contains spills and drips. • Always bond metal containers to metal receivers when transferring large volumes of flammable liquids or gases. • Static electricity can ignite flammable gases or vapors. If static electricity is a problem, minimize static electricity by spraying with an antistatic agent. Use nonconductive materials (floors, mats, etc.) and grounding straps on instruments and machines, especially when transferring flammable chemicals between metal containers. These reduce the risk of generating static sparks. The greatest hazard from static electricity is in the winter when the air is dry. • Never heat flammable chemicals with an open flame, use a water bath, oil bath, heating mantle, hot air bath, etc. • Use a fume hood when there is a possibility of dangerous vapors. • Cold rooms pose a unique set of problems. One big problem with anything stored in a walk- in cold room is that outside (hallway or room) air brings in moisture which condenses on everything that is cold. This will lead to mold which thrives on paper and glue of labels and can make stored containers "unknowns." Take precautions when storing flammable chemicals in a refrigerator. Refrigerator temperatures are almost always higher than the flash points of flammable liquids. Compressors and circuits are often located at the bottom of the refrigerator where vapors from small spills or leaks can accumulate. Electrical sparks from a conventional refrigerator can then ignite the

48 flammable vapors that build up inside. Unless a cold room is ventilated and has a fire suppression sprinkler system, do not store flammable liquids there. Two kinds of refrigerators are approved for storage of flammables: 1. Flammable liquid storage refrigerators. These have no spark sources within the refrigerator cabinet. There are, however, spark sources outside the refrigerator cabinet from switches, motors, relays, etc. These spark sources can ignite flammable vapors present outside of the refrigerator. A bottle of flammable liquid that drops and breaks near one of these refrigerators can easily be ignited by the sparks. 2. Explosion-proof refrigerators. These refrigerators are considerably more expensive because they have all spark sources completely sealed inside and are safe for flammable atmospheres both within and outside of the refrigerator cabinet. In certain cases a conventional refrigerator can be modified by the Facilities Management Electric Shop to become a flammable liquid storage refrigerator. Only those refrigerators that do not have a frost-free feature can be modified in this way. A frost-free refrigerator incorporates heated coils and fans that cannot be removed from the cabinet. Conventional refrigerators in laboratories and cold rooms are not safe for flammable storage and must be labeled " NO FLAMMABLES ". Labels are available from the EHS Department.

Moving Chemicals A chemical sitting on a shelf in its original container presents less hazard than when that same container is moved. Whenever a chemical is moved there is a risk of the container breaking resulting in an uncontrolled release. Many of the campus calls for emergency responders are the result of chemicals spilled while moving them within a building. Whether you are transporting chemicals across your lab or across the campus, take precautions. Use secondary containment. No matter how careful you are, containers can drop and bottles can break. An unprotected chemical container breaking in an elevator could be disastrous. Good secondary containment can mean the difference between a small inconvenience and a major building evacuation. Secondary containment is defined as a container capable of containing the entire contents of the original container in the event of a spill. Typical secondary containment vessels include plastic trays made of chemically resistant materials such as Nalgene. Extra precautions for vehicles. The transportation of chemicals in vehicles on public roads presents additional safety and legal problems. A container of flammable solvent or toxic material ruptured in a road accident drastically increases the risk to your health and makes rescue difficult. Chemicals should never be transported in the passenger compartment of a vehicle. The Department of Transportation (DOT) regulates the transportation of hazardous materials on public roads. Depending on the type and quantity of material transported, the person driving may be required by law to have a special (e.g., commercial) driver's license, carry proper shipping papers and use specified packaging. If you must transport hazardous chemicals on public roads, call EHS first. We can give you guidance on how to do it safely. In some cases we may transport your materials for you.

49 Shipping Hazardous Materials. Many different government agencies regulate hazardous materials. If a hazardous substance is to be sent or transported off campus (e.g., FedEx, etc.), Department of Transportation (DOT) rules and regulations apply. The International Air Transport Association (IATA) sets rules for air transport of hazardous materials. You must attend a training class and be certified to ship (i.e., give to a commercial carrier like FedEx) any hazardous material. EHS can provide a training class which will certify you for shipping chemicals or biological materials. Chemical Disposal

Disposal of waste and unwanted chemicals has become increasingly complicated. The U.S. Environmental Protection Agency (EPA) and the South Dakota Department of Environment and Natural Resources (DENR) regulate the disposal and treatment of hazardous waste, including waste laboratory chemicals. There are many chemicals that can be properly disposed of by discharge into the sanitary sewer after elementary neutralization or other chemical treatment method, by discharge into the sanitary sewer with no neutralization or other chemical treatment required, or directly into the normal trash. No laboratory chemicals or chemical wastes may be disposed of in any campus incinerator. Campus incinerators do not meet the destruction standards for chemical waste, and are not permitted for chemical waste disposal. Label Your Waste . Regardless of the disposal route, all containers of hazardous material must be labeled with their contents. Labeling lab generated waste material as “Solid Waste” is not really helpful; we can observe both that it is solid (or not) and that no one wants it. What is needed is the hazardous ingredient (i.e., the reason it is hazardous and not just laboratory trash) and any other major components. For sample collections of synthesis intermediates, extracted substances and collections of chemically similar materials of great variety and small amounts (i.e., dyes and stains), keep these together, well capped vials placed upright in a box, not as a pile in a bucket or in a sharps container. This is the best way to be able to present the collection to the people who receive chemicals for ultimate disposal without having to write down each and every name (e.g., they can be named as a group). With chemicals produced in a combinational array, this will be helpful as well; keep similar series of different variants together as a neatly arranged group in a box. Unknowns . Analysis and disposal of material for which the identity is not known can be expensive, from $30 to $1500 per unknown. Consult with other workers in the area who may have an idea as to the identity of the material. Even a general chemical classification (such as aromatic sulfur compounds) can be very helpful. A phone call to a colleague who has left will pay for itself. To prevent unknowns, remember to label all your containers. Suitable Containers . Chemical mixtures, aqueous solutions, other liquids and reaction products should be placed in a suitable container. Empty containers in which the chemicals are supplied are usually satisfactory for removal. Make sure all containers are tightly closed and contain the material that they hold. Insure any waste reaction mixture or cleaning solution is done reacting and no longer producing a gas prior to securely capping, overpressure may produce a spill or explosion. Where does it go? When the EHS Department removes chemicals from your laboratory, it is first brought to a campus storage facility. There, the chemicals are sorted. Chemical waste that

50 cannot be managed on campus is shipped out of state by a commercial hazardous waste vendor. The normal disposal is via incineration. Incineration of hazardous chemical waste is required to achieve a 99.99% destruction efficiency and many chemicals are destroyed at even greater efficiency. Toxic metals are removed from the exhaust gases and remain in the ash. Although incineration is a superior method of hazardous waste disposal, all incinerators emit carbon dioxide (a global greenhouse gas) and other products of combustion, some of which may be toxic. No disposal method is without risk.

51

Biological Safety

The hazards posed by biological materials, plus the risks of infections resulting from exposure to infectious materials, are important considerations when working in a biological laboratory. Several deaths have resulted from infections acquired in labs where etiological agents have been in use. Controlling exposures (and the resulting infections) requires an understanding of the factors involved in disease transmission in the laboratory. The most common routes of exposure are ingestion, inhalation, and self-inoculation. The development of an infection subsequent to an exposure to an infectious agent depends upon individual susceptibility, the size of the dose, and the pathogenicity of the organism. The only one of these three factors within the control of the investigator is the size of the dose. If all exposures can be kept below the infectious dose, the risk of infection is greatly minimized. This is the basis for safety in the biological laboratory. References which expand on the content of this section include: • The CDC/NIH publication Biosafety in the Microbiological and Biomedical Laboratories (BMBL ), 5th Edition http://www.cdc.gov/biosafety/publications/bmbl5/index.htm • Primary Containment for Biohazards: Selection, Installation and Use of Biosafety Cabinets , 3rd Edition ( http://www.cdc.gov/od/ohs/biosfty/primary_containment_for_biohazards.pdf ) • The American Public Health Association publication Control of Communicable Diseases , 19th Edition, David L. Heymann, Editor • The American Industrial Hygiene Association publication Biohazards Reference Manual , 2 nd Edition • National Research Council publication Biosafety in the Laboratory: Prudent Practices for the handling and disposal of Infectious Materials

General Precautions Employees who work with or around an agent for which there is a vaccine should discuss with their PI / supervisor pros and cons about immunization for that particular agent. Please inform the Director, Environmental Health & Safety (EHS)and the Chair, USD Institutional Biosafety Committee (IBC) of the receipt of any material classified as Biosafety Level 2 (BSL2) or above; include information about the location, storage, use, precautions, and emergency procedures. Use a biohazard warning symbol to designate labs or storage areas housing human blood, blood products, or tissues, and any pathogenic agents. If a laboratory is conducting work at BSL2 level or above, a warning sign identifying the Risk Group for the agent. Emergency contact information and any special precautions required are usually on file with

52 USD Public Safety and Lee Medical School administration. Appendix C is a summary of biosafety levels and corresponding agents. The receipt of any material deemed by the CDC/USDA as a Select Agent requires prior approval. Currently the USD is not registered to work with such agents, contact the Director, EHS and the Chair, USD IBC if you are considering such work. Work with BSL2 Agents or above require submitting a protocol for approval with the Institutional Biosafety Committee. USD requires use of the rDNA, transgenic animal and biological agent registration form, which is forwarded to the USD IBC for review and approval. Basic precautions while working in a biological laboratory • Do not eat, drink, apply cosmetics or lip balm or store food in the laboratory. • Never mouth pipette. • Wear safety eyewear and/or face shields, if appropriate, when working in a laboratory. • Wear disposable, high cuff, latex or nitrile gloves when working with biohazards. Remember, latex gloves are permeable to organic solvents, including ethanol. See Appendix A for an example glove chart. • Thin gloves offer little protection against cuts, bites, scratches, etc. Use the thickest gloves allowed by your work but do not sacrifice the dexterity required by your work. • Wearing two pairs of thinner gloves will permit the safe removal of the outer pair in case of inadvertent contamination. • Wear a lab coat while in the laboratory. The lab coat should be buttoned and, if working with Biological agents, the sleeves tucked into the gloves or otherwise restrained. • Disposable Tyvek lab coats are available and recommended for work conducted in biosafety cabinets or during animal surgeries. • Do not wear lab coats outside of the laboratory (e.g. breakroom, conference rooms, or outside the floor or building) • Use at least a Class II biosafety cabinet for work with biohazardous materials. • Procedures which might generate aerosols should be performed in the rear third (i.e., toward the back) of the biosafety cabinet. • Avoid the use of needles, scalpels, or other sharp instruments whenever possible. If needles and syringes must be used, to avoid aerosols, cover the tip with absorbent material when adjusting the volume or withdrawing the tip from a septum or injection site. • Dispose sharps in a puncture resistant container. Do not re-sheath or remove used needles; insert the whole assembly into the container. • Wash any puncture or stick with soap and water and report it to your supervisor and EHS. • If experimentation requires the use of pathogens, first develop and test all procedures using nonpathogenic agents (i.e. perform “dry runs”). • Use disposable glass or plastic-ware. If non-disposable glassware must be used, disinfect contaminated items before cleaning. • Clean biological spills immediately with a fresh solution of chlorine bleach diluted 1:10 with water. • All liquid waste materials must be decontaminated / deactivated before disposal. • Discard non-sharp disposable materials (e.g., gloves, pipettes, pipette tips, and plastic tubes) that are exposed to potentially infectious materials in polyethylene biohazard bags, for transport by Medical Waste Transport, Inc. • Treat blood and other potentially infectious fluids with 10% chlorine bleach and decant down the drain.

53 • Do not dispose of blood or sharps with the regular laboratory trash. Human Blood, Blood Products, Secretions or Other Potentially Infectious Materials Persons who work with blood or blood products are at increased risk of contracting hepatitis. Hepatitis B vaccination is recommended for any individual working with blood or potentially HBV containing material. OSHA requires all potential HBV exposed personnel to have either an HBV vaccination, or a signed declination on file. The most important way for personnel to protect themselves from Hepatitis B, as well as other bloodborne infections, is to follow universal precautions and handling all blood, blood products, secretions and other potentially infectious material as if infectious. If an exposure to other potentially infectious material (OPIM) occurs, report it immediately to your supervisor and the Director, EHS for evaluation and possible treatment. The USD Bloodbourne Pathogen Plan contains the USD program. Recombinant DNA Experiments If you plan to conduct experiments involving recombinant DNA (rDNA), be aware that although the IBC does recognize the exemptions detailed in the NIH rDNA regulations, USD requires that the exemption be reviewed by the USD IBC. Any recombinant DNA work must seek IBC approval prior to initiation of work. Contact the IBC for the proper application forms. Work With Potentially Infectious Materials For most biohazardous agents, the routes of potential infection are inoculation, ingestion and inhalation. Wearing gloves, a lab coat, safety glasses, and using a biological safety cabinet, if appropriate, greatly minimizes an individual’s exposure. Decontamination of wastes can be accomplished for all these agents by 30-minute exposure to fresh 10% chlorine bleach. Note that the color change associated with oxidation of media is not a good indicator of inactivation. All human specimens should be regarded and handled as infectious. The risk from human specimens is not restricted to hepatitis or AIDS, but includes many others that may be found in blood, blood products, urine, feces, amniotic fluid, etc. Researchers occasionally receive blood that has been designated “not for transfusion” and other fresh specimens that have not been screened for these agents. Research with human specimens is generally BSL 2, and the procedures set forth by the CDC/NIH for those biosafety levels should be followed. Cell Lines While information on specific cell lines is not included, it is important to recognize there is no “normal” cell line; many reputedly “normal” lines harbor viruses and potentially hazardous gene sequences. Handle these materials as if infectious and decontaminate culture wastes prior to disposal. All cells should be fixed before subjecting them to an aerosol generating process (e.g., flow cytometers). Viruses Fluids, tissues, isolates and cell cultures containing infectious viruses pose a risk following exposure by ingestion, percutaneous or parenteral inoculation, and droplet or aerosol contamination of the mucous membranes of the eyes, nose, mouth, or broken skin. The aerosol risk from handling large volumes and concentrated stocks is great since some viruses are stable at ambient temperatures and withstand drying.

54 Variation in viral structures results in differential susceptibility to “germicidal” agents and detergents; however, chlorine bleach treatment is usually effective. See the reference material at the end of this section for a chart showing the relative risk from oncogenic viruses. Bacteria Many bacteria are ubiquitous, but some of these such as Staphylococcus aureus and group A streptococci, are responsible for serious infections in man. The potential routes of exposure are as discussed above for viruses. Aerosols are of major concern when working with large volumes or concentrated stocks and with pathogenic spore forming species since spores resist adverse or extreme conditions. Safety glasses must be worn when handling bacteria, especially those that infect the conjunctiva, e.g., N. gonorrhea. Work at a higher biosafety level is required when large volumes, highly concentrated stocks, or aerosol generating procedures are employed with infectious bacteria. All wastes must be decontaminated prior to disposal; autoclaving or chlorine bleach treatments are effective. Parasites Infectious stages of protozoal parasites of humans may be present in blood, feces, lesion exudates, and infected arthropods. Depending on the parasite, accidental parenteral inoculation, transmission by arthropod vectors, skin penetration (including bites from infected animals), and ingestion are the primary laboratory hazards. Aerosol or droplet exposures of the mucous membranes of the eyes, nose, or mouth with trophozoites are potential hazards when working with cultures of Leishmania and Trypanosoma species. All exposures should be reported to the Principal Investigator and treated immediately, e.g., wipe bite with 70% ethanol or irrigate eye with distilled water. In general, protozoa are very fragile, sensitive to drying, and, with notable exceptions such as T. cruzi, lysed even by water; however, all spills and waste must be actively treated. BSL2 containment procedures are recommended for work with all parasites except Babesia, which require BSL3 containment procedures. Fungi Fungi are generally not significant causes of human disease. Transmission of fungal diseases from person to person is extremely rare. Fungal spores however are generally very allergenic and some of the fungal constituents and by-products can be highly toxic, such as aflatoxin B. The more common hazardous fungi used in laboratories include: Blastomyces dermatitides, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, and Sporothrix schenckii. ll of these agents should be handled at BSL2 levels. A classification of microorganisms according to hazards is presented in Appendix B of the Guidelines for Recombinant DNA Research, available from the Chair, IBC. Note that agents of class 1-4 should be handled according to the corresponding biosafety containment level and that there are restrictions against importation of class 5 agents. Work with Laboratory Animals

55 All vertebrate animal experimentation requires the approval of the USD IACUC. Animals are to be housed only in accredited animal facilities and are not to be kept in laboratories for more than 24 hours. Users of laboratory animals must recognize that virtually all laboratory animal species can carry pathogens that are infectious to humans. Inoculated animals readily transmit viruses to cage mates by inhalation and contact with urine, feces, sputum, etc. Caution should be taken when working with any animal. All urine, feces or blood should be cleaned up as soon as practical. Concern for the health of others who do not work directly with animals should be paramount when laboratory animals are transported or used in general laboratory areas outside of an animal facility. Registration of transgenic animals with the USD IACUC is required. Zoonoses Although animal diseases do not commonly affect humans, some few do. A few of the more important animal reservoirs are: • Primates . Diseases such as tuberculosis, shigella, campylobacter and salmonella can be serious health threats. Herpesvirus B carried by rhesus, cynomolgus, and other Old World monkeys can cause fatal encephalitis in man. • Dogs and Cats . Bite wound infections, cat scratch disease, visceral larval migrans and sarcoptic mange from dogs and toxoplasmosis and fungi’s (such as ringworm) from cats are common. • Rodents . Precautions should be taken against toxoplasmosis, lymphocytic choriomeningitis, salmonella, shigella, and ringworm. Toxoplasmosis is one of the most commonly acquired parasitic diseases in the laboratory. • Rabbits , Sheep, Swine , and Birds can be the source of tularemia, Q fever, Erysipelas, and Chlamydia (psittacosis), respectively. Some requirements for the use of animals: • Protocols involving the use of hazardous chemicals or biohazardous agents in animals must be reviewed with the IBC, and Director, EHS prior to initiation. Use of radioisotopes also requires registration of the principle investigator and his staff by the Director, EHS (see the USD Radiation Safety Policy). • All users of laboratory animals must have an active tetanus immunization and other immunizations as appropriate, e.g., rabies, HBV. • Bites or scratches that break the skin should be washed thoroughly with soap and water and reported to your supervisor, and the Director, EHS. • Wearing a facemask, gloves and a lab coat is required of users of animals to reduce aerosol, direct contact or inadvertent oral and nasal contact with contaminated materials. • A full-face respirator is required for those at high risk. • Lab coats should be changed and hands thoroughly washed if an animal, its fluids, or feces is touched. Tyvek lab coats available. • Allergic responses to laboratory animals are the most common cause of human disease related to the use of animals in research. Allergy results from the direct or indirect exposure to allergens such as skin contact or inhalation of fur, dander, saliva, urine, serum, etc. Symptoms can vary from wheezing, sneezing and rhinitis to itching eyes and skin, obvious rashes, and asthma. Do not ignore the symptoms. Continued exposure can lead to anaphylaxis and is life threatening. • Pregnant employees should not expose themselves to feces, dander, or biohazard areas, and should suspend work involving the handling of cats and monkeys. Likewise, pregnant

56 women without immunity to toxoplasmosis should avoid cat contact to avoid the possibility of congenital disease and fetal death. Aerosol-Generating Processes

Aerosols (dispersions of particles in air) can result from the use of blenders, mixers, sonicators, cell disrupters, centrifuges, syringes, pipettes, aspirators, test and centrifuge tube caps. Several well-documented studies have made it clear that great attention must be given to prevent contamination of room air with the suspension of liquid or solid particles containing hazardous materials including radioisotopes, infectious agents (viruses and mycoplasma from “normal” cells), as well as toxic chemicals and carcinogens. Particle size is a factor in determining the path an aerosol will follow. Particles in the range of 1 to 5 microns present the greatest hazard to the laboratory worker, because they more readily penetrate the respiratory tract than larger particles and are more readily retained than smaller or larger particles. Many laboratory procedures produce aerosols with particles in this range. Particles larger than 10 microns fall out on surfaces or are impinged on materials with an opposite electrostatic charge. In the respiratory tract, larger particles do not penetrate into the lower spaces but are removed by interception and impaction in the upper respiratory tract and subsequently expelled or swallowed. Large droplets that fall out on surfaces dry quickly and secondary aerosols of the dry particles can be created by air currents or laboratory activity. Significant settling of larger particles from an aerosol can occur in five minutes; however, most of the remaining small particles require 30 minutes to an hour to settle, assuming that fresh currents of air do not prevent their settling. For that reason, it is best to wait before cleaning up a spill of infectious virus or other biologically hazardous material. Besides the direct effects of aerosols, they may also contaminate surfaces of the skin or equipment and subsequently enter the body by hand-to- mouth contact and ingestion or through abrasions of the skin. In addition to avoiding the creation of an aerosol, three general approaches are recommended to decrease the hazards of aerosols associated with research on tumor specimens, cell and virus cultures and concentrates, and toxic chemical materials: • Reduce the extent or concentration of the aerosol. • Contain the aerosol in a primary barrier system. • Use personal respiratory protection and protective laboratory clothing. The following is an example of some aerosol generating activities: • Forced expulsion of the last drop of liquid or mixing of liquid by alternately sucking and blowing with the pipette, creating splashes and bubbles. • Removing the cap or stopper from bottle after vigorous shaking to mix, wash or re-suspend material. • Blending materials to disrupt cells, release enzymes or viruses, to homogenize suspensions, etc., without aerosol tight cover seals or leak-proof rotor bearings. • Sonic disruption of cells or organelles. • Grinding tissue with mortar and pestle, glass tissue grinder or ball mill. • Pouring hazardous materials from one container to another, e.g., decanting supernatants. • Sterilizing a wire loop or needle in a flame, creating splatter.

57 • Withdrawing a syringe needle as from a vaccine bottle or following inoculation of experimental animals. • Weighing dry hazardous materials. • Opening a freeze-dried preparation. • Removing plugs from flasks and tubes. • Handling cages that held infected animals or large animals in open areas or unventilated cages. Recommended Measures to Decrease Hazards from Aerosols • Use gravity flow of liquid with pipette calibrated for mark-to-mark drain-to-tip delivery and with pipette tip in contact with container wall. • Use swirling motion rather than shaking, allow aerosol to settle for a few minutes after bubbles disappear before removing cap or stopper. • Use special safety containers with seals to prevent escape of aerosols; use drain/siphon system to remove contents without removing cover. • Use cup or chamber that is aerosol tight; allow aerosol to settle before opening cup. • Place sonicator in fume hood or laminar flow cabinet. Use slow speeds; use a clear plastic or inflatable glove bag to further contain the operation within the safety cabinet; allow aerosol to settle before removing cover. • Use transfer pipettes or closed siphon or vacuum technique. • Gradually dry loop or needle near flame, or use specially designed incinerator for loops and needles. • Use sterile cotton gauze to enclose needle; if experiment permits, use disinfectant with cotton or gauze. • Use draft-free, low-humidity enclosure for balance; discharge static electricity; use tared weighing containers not open weighing dishes or papers. • If material is in an ampoule, nick the ampoule with a file, cover its neck with sterile gauze. If material is in a rubber-stoppered bottle, first relieve vacuum with a hypodermic needle. • If material is to be dissolved or suspended in liquid, introduce the liquid with a syringe and cover needle with gauze wetted with disinfectant. • Avoid disturbing cage contents; if animals are held in open areas, use liquid disinfectants during cage cleaning; keep area clean; use personal respirator. Biological Safety Cabinets (Tissue Culture Hoods)

BSCs are only one part of an overall biosafety program. Detailed descriptions of acceptable work practices, procedures, and facilities, described as biosafety levels 1 through 4, are presented in the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories , 5th edition (BMBL). The following information on BSCs was excerpted from the NSF/ANSI publication Class II Biosafety Cabinetry (NSF 49-2002). BSCs are designed to provide personnel, environmental and product protection when appropriate practices and procedures are followed. Three kinds of biological safety cabinets, designated as Class I, II and III have been developed to meet varying research and clinical needs. The similarities and differences in protection offered by the various classes of biosafety cabinets are reflected in the following table:

58 Biological Risk Protect Protect Protect Product BSC Class Assessed Personnel Environment` BSL 1 -3 Yes No Yes I BSL 1 -3 Yes Yes Yes II (A, B1, B2, B3) BSL 4 Yes Yes Yes III B1, B2 The Class I BSC The Class I BSC provides personnel and environmental protection, but no product protection. It is similar to a chemical fume hood, but has a HEPA filter in the exhaust system to protect the environment. In the Class I BSC, unfiltered room air is drawn across the work surface. Personnel protection is provided by this inward airflow as long as a minimum velocity of 75 linear feet per minute (lfpm) is maintained through the front opening. With the product protection provided by the Class II BSCs, general usage of the Class I BSC has declined. However, in many cases Class I BSCs are used specifically to enclose equipment (e.g., centrifuges, harvesting equipment or small fermenters), or procedures (e.g., cage dumping, aerating cultures or homogenizing tissues) with a potential to generate aerosols. HEPA filters remove particles equal to, greater than, and less than 0.3 µm (i.e., practically includes all bacteria, spores and viruses) with an efficiency of 99.97%. A detailed explanation of HEPA filter efficiency and the mechanics of particle collection have been well documented. The Class I BSC is hard-ducted to the building exhaust system, and the building exhaust fan provides the negative pressure necessary to draw room air into the cabinet. Cabinet air is drawn through a HEPA filter as it enters the exhaust plenum. A second HEPA filter may be installed at the terminal end of the exhaust. Some Class I BSCs are equipped with an integral exhaust blower; the cabinet blower must be interlocked with the building exhaust fan. In the event that the building exhaust fan fails, the cabinet exhaust blower must turn off so that the exhaust ducts are not pressurized. Filters should be installed on the intake side of the fan. Also, note that use of two filters increases the static pressure on the fan. If the ducts are pressurized and the HEPA filter develops a leak, contaminated air could be discharged into other parts of the building or the environment. A steel panel with armholes to allow access to the work surface can be added to the Class I cabinet. The restricted opening results in increased inward air velocity, thereby increasing worker protection. For added safety, arm-length gloves can be attached to the paned. Makeup air is then drawn through an auxiliary air supply opening (which may contain a filter) and/or around a loose-fitting front panel.

59 The Class II BSC The Class II (Types A1, A2 (formerly B3), B1, B2) biological safety cabinets provide personnel, environmental and product protection. Airflow is drawn around the operator into the front grille of the cabinet, which provides personnel protection. In addition, the downward laminar flow of HEPA-filtered air provides product protection by minimizing the chance of cross- contamination along the work surface of the cabinet. Because cabinet air has passed through the exhaust HEPA filter, it is contaminant-free (environmental protection), and may be recirculated back into the laboratory (Type A BSC) or ducted out of the building (Type B BSC). HEPA filters are effective at trapping particulates and infectious agents, but not at capturing volatile chemicals or gases. Only BSCs that are ducted to the outside may be used when working with volatile toxic chemicals. All Class II cabinets are designed for work involving microorganisms assigned to biosafety levels 1, 2 and 3. Class II cabinets provide the microbe-free work environment necessary for cell culture propagation, and also may be used for the formulation of nonvolatile antineoplastic or chemotherapeutic drugs. The Class II, Type A1 BSC This class was formerly known as Class II, Type A. An internal blower draws sufficient room air through a front grille to maintain a minimum inflow velocity of at least 75 lfpm at the face opening of the cabinet. The supply air flows through a HEPA filter and provides particulate-free air to the work surface. Laminar airflow reduces turbulence in the work zone and minimizes the potential for cross-contamination. The downward moving air “splits” as it approaches the work surface; the blower draws part of the air to the front grille and the remainder to the rear grille. Although there are variations among different cabinets, this split generally occurs about halfway between the front and rear grilles, and two to six inches above the work surface. The air is then discharged through the rear plenum into the space between the supply and exhaust filters located at the top of the cabinet. Due to the relative size of these two filters, approximately 30% of the air passes through the exhaust HEPA filter and 70% recirculates through the supply HEPA filter back into the work zone. Most Class II, Type A cabinets has dampers to modulate this 30/70 division of airflow. An un-ducted Class II Type A1 BSC is not to be used for work involving volatile or toxic chemicals. The buildup of chemical vapors in the cabinet (by recirculated air) and in the laboratory (from exhaust air) could create health and safety hazards. It is possible to duct the exhaust from a Type A cabinet out of the building. However, it must be done in a manner that does not alter the balance of the cabinet exhaust system, thereby

60 disturbing the internal cabinet airflow. The typical method of ducting a Type A1 cabinet is to use a “thimble”, or canopy hood, which maintains a small opening (usually 1 inch) around the cabinet exhaust filter housing. The volume of the exhaust must be sufficient to maintain the flow of room air into the space between the thimble unit and the filter housing (contact manufacturers for any additional specifications). The thimble must be removable or be designed to allow for operational testing of the cabinet. The performance of a cabinet with this exhaust configuration is unaffected by fluctuations in the building exhaust system. “Hard-ducting” (i.e., direct connection) of Class II Type A1 cabinets to the building exhaust system is not recommended. The building exhaust system must be precisely matched to the airflow from the cabinet in both volume and static pressure. However, fluctuations in air volume and pressure that are common to all building exhaust systems make it difficult, if not impossible, to match the airflow requirements of the cabinet. The Class II, Type A2 BSC Formerly known as the Class II Type B3. This biological safety cabinet is typically a ducted Type A cabinet having a minimum inward airflow of 100 lfpm, although it may be exhausted into the lab space. All positive pressure contaminated plenums within the cabinet are surrounded by a negative air pressure plenum. Thus, leakage in a contaminated plenum will be into the cabinet and not into the environment. A connection to the building exhaust system required. The Class II, Type B1 BSC Room air is drawn through the face opening of the cabinet at a minimum inflow velocity of 100 lfpm. As with the Type A cabinet, there is a split in the down- flowing air stream just above the work surface. In the Type B cabinet, approximately 70 percent of the down flow air exits through the rear grille, passes through the exhaust HEPA filter, and is discharged from the building. The remaining 30 percent of the down flow air is drawn through the front grille. Since the air which flows to the rear grille is discharged into the exhaust system, activities that may generate hazardous chemical vapors or particulates should be conducted towards the rear of the cabinet. Type B1 cabinets must be hard-ducted, preferably to their own dedicated exhaust system, or to a properly designed laboratory building exhaust. As indicated earlier, blowers on laboratory exhaust systems should be located at the terminal end of the ductwork. A failure in the building exhaust system may not be apparent to the user, as the supply blowers in the cabinet will continue to operate. A pressure-independent monitor should be installed to sound an alarm

61 and shut off the BSC supply fan, should failure in exhaust airflow occur. Since not all cabinet manufacturers supply this feature, it is prudent to install a sensor in the exhaust system as necessary. To maintain critical operations, laboratories using Type B BSCs should connect the exhaust blower to the emergency power supply. The Class II, Type B2 BSC This BSC is a total-exhaust cabinet; no air is recirculated within it. This cabinet provides simultaneous primary biological and chemical containment. The supply blower draws in room air or outside air at the top of the cabinet, passes it through a HEPA filter and down into the work area of the cabinet. The building or cabinet exhaust system draws air through both the rear and front grilles, capturing the supply air plus the additional amount of room air needed to produce a minimum calculated or measured inflow face velocity of 100 lfpm. All air entering this cabinet is exhausted, and passes through a HEPA filter (and perhaps some other air-cleaning device such as a carbon filter) prior to discharge to the outside. Exhausting as much as 1200 cubic feet per minute of conditioned room air makes this cabinet expensive to operate. Should the building or cabinet exhaust fail, the cabinet will be pressurized, resulting in a flow of air from the work area back into the laboratory. Cabinets built since the early 1980’s usually have an interlock system installed by the manufacturer to prevent the supply blower from cease operating whenever the exhaust flow is insufficient. Presence of such an interlock system should be verified; systems can be retrofitted if necessary. A pressure-independent device should monitor exhaust air movement. Special Applications Class II BSCs can be modified to accommodate special tasks. For example, the front sash can be modified by the manufacturer to accommodate the eyepieces of a microscope, or the work surface can be designed to accept a carboy, a centrifuge, or other equipment that requires containment. A rigid plate with armholes can be added if needed. Good cabinet design, microbiological aerosol tracer testing of the modification, and appropriate certification are required to ensure that the basic systems operate

62 properly after modification. Maximum containment potential is achieved only through strict adherence to proper practices and procedures. The Class III BSC The Class III biological safety cabinet was designed for work with biosafety level 4 microbiological agents, and provides maximum protection to the environment and the worker. It is a gas-tight enclosure with a non-opening view window. Access for passage of materials into the cabinet is through a dunk tank (that is accessible through the cabinet floor) or double-door pass-through box (such as an autoclave) that can be decontaminated between uses. Reversing that process allows for safe removal of materials from the Class III biosafety cabinet. Both supply and exhaust air are HEPA filtered. Exhaust air must pass through two HEPA filters, or a HEPA filter and an air incinerator, before discharge to the outdoors. Airflow is maintained by a dedicated independent exhaust system exterior to the cabinet, which keeps the cabinet under negative pressure (usually about 0.5 inches of water pressure). A connection to the building exhaust system required. Long, heavy-duty rubber gloves are attached in a gas-tight manner to ports in the cabinet and allow for manipulation of the materials isolated inside. Although these gloves restrict movement, they prevent the user’s direct contact with the hazardous materials. The trade-off is clearly on the side of maximizing personal safety. Depending on the design of the cabinet, the supply HEPA filter provides particulate-free, albeit somewhat turbulent, airflow within the work environment. Several Class III cabinets can be joined together in a “line” to provide a larger work area. Such cabinet lines are custom-built; the equipment installed within the cabinet line (e.g., refrigerators, small elevators, shelves to hold small animal cage racks, microscopes, centrifuges, incubators, etc.) is generally custom-built as well. Furthermore, Class III cabinets are usually only installed in maximum containment laboratories that have controlled access and require special ventilation or other support systems. Laminar flow “clean benches” Laminar flow clean air benches (horizontal or vertical) are not BSCs. These benches should never be used when handling cell culture materials or drug formulations, or when manipulating potentially infectious materials. The worker can be exposed to materials (including proteinaceous antigens) being manipulated on the clean bench, which may cause

63 hypersensitivity. These devices only provide product protection. They can be used for certain clean activities, such as the dust-free assembly of sterile equipment or electronic devices. Horizontal units discharge HEPA-filtered air across the work surface and toward the user. Horizontal clean air benches should never be used as a substitute for a biological safety cabinet in research, biomedical, or veterinary laboratories. Vertical laminar flow clean benches may be useful in pharmacies when a clean area is needed for preparation of intravenous drugs. While these units generally have a sash, the air is usually discharged into the room under the sash, resulting in the same potential problems as the horizontal laminar flow clean benches. Chemicals in BSCS Work with infectious microorganisms often requires the use of various chemical agents, and many commonly used chemicals vaporize easily. Therefore, evaluation of the inherent hazards of the chemicals must be part of the risk assessment when selecting a BSC. Volatile or toxic chemicals should not be used in un-ducted Class II, Type A1 cabinets since vapor buildup inside the cabinet presents a fire hazard. In order to determine the greatest chemical concentration that might be entrained in the air stream following an accident or spill, it is necessary to evaluate the quantities to be used. The electrical systems of Class II cabinets are not spark-proof, so no chemical concentration should be allowed that would approach the lower explosive limits of the compound. Furthermore, since Class II, Type A1 cabinets return chemical vapors to the cabinet workspace and the room; they may expose the operator and other room occupants to toxic chemical vapors. Rather than a BSC, consider using a chemical fume hood, which is designed for work with volatile chemicals. Chemical fume hoods are connected to the building exhaust system and operate with single-pass air ducted directly outside the building. They also are used when manipulating chemical carcinogens. However, because they also are ducted to the outside, Class I and Class II, Type B2 biological safety cabinets can be used when manipulating small quantities of volatile chemicals as an adjunct to microbiological studies. The Class II, Type B1 cabinet also may be used with minute or tracer quantities of nonvolatile chemicals. Many virology and cell culture laboratories use diluted preparations of chemical carcinogens and other toxic substances. Prior to maintenance, careful evaluation must be made of potential problems associated with decontaminating the cabinet and the exhaust system. Air treatment systems, such as a charcoal filter in a bag-in/bag-out housing, may be required so that effluents meet applicable emission regulations. Radiological Hazards in the BSC Volatile radionuclides such as 125 I should not be used within Class II, Type A cabinets. When using nonvolatile radionuclides inside a BSC, the same potential inherent hazards exist as when working with radioactive materials on the bench top.

64 Work that has the potential for splatter or aerosolization can be done within the BSC, and monitoring for radioactivity must be done. BSCs should be decontaminated as needed. A straight vertical (not sloping) beta shield may be used inside the BSC to provide worker protection when appropriate. BSC Use: Work Practices and Procedures

Preparing for Work within a Class II BSC Preparing a written checklist of materials necessary for a particular activity and placing necessary materials in the BSC before beginning work serves to minimize the number of arm- movement disruptions across the fragile air barrier of the cabinet. The rapid movement of a worker’s arms in a sweeping motion into and out of the cabinet will disrupt the air curtain and may compromise the partial barrier containment provided by the BSC. Moving arms in and out slowly, perpendicular to the face opening of the cabinet will reduce this risk. Other personnel activities in the room (e.g., rapid movement, open/closing room doors, etc.) may also disrupt the cabinet air barrier. Laboratory coats must be worn buttoned over street clothing; latex gloves are worn to provide hand protection. A solid front, back-closing lab gown provides better protection of personal clothing than a traditional lab coat. Gloves should be pulled over the knitted wrists of the gown, rather than worn inside. Elasticized sleeves can also be worn to protect the investigator’s wrists. Before beginning work, the user should adjust the stool height so that his/her face is above the front opening. Manipulation of materials should be delayed for approximately one minute after placing the hands/arms inside the cabinet. This allows the cabinet to stabilize and to “air sweep” the hands and arms to remove surface microbial contaminants. When the user’s arms rest flatly across the front grille, room air may flow directly into the work area, rather than being drawn through the front grille. Raising the arms slightly will alleviate this problem. The front grille must not be blocked with research notes, discarded plastic wrappers, pipetting devices, etc. All operations should be performed at least four “4” inches from the front grille on the work surface. Materials or equipment placed inside the cabinet may cause disruption to the airflow, resulting in turbulence, possible cross-contamination, and/or breach of containment. Extra supplies (e.g., additional gloves, culture plates or fl asks, culture media) should be stored outside the cabinet. Only the materials and equipment required for the immediate work should be placed in the BSC. BSCs are designed to be operated 24 hours per day, and some investigators find that continuous operation helps to control the laboratory’s level of dust and other airborne particulates. Although energy conservation may suggest BSC operation only when needed, especially if the cabinet is not used routinely, room air balance is an overriding consideration. In some instances, room exhaust is balanced to include air discharged through ducted BSCs. Cabinet blowers should be operated at least three to five minutes before beginning work to allow the cabinet to “purge”. This purge will remove any particulates in the cabinet. The work surface, the interior walls (not including the supply filter diffuser), and the interior surface of the window should be wiped with 70% ethanol (EtOH), a 1:100 dilution of household bleach (i.e., 0.05% sodium hypochlorite), or other disinfectant as determined by the investigator to meet the

65 requirements of the particular activity. When bleach is used, a second wiping with sterile water is needed to remove the residual chlorine, which may eventually corrode stainless steel surfaces. Wiping with non-sterile water may recontaminate cabinet surfaces, a critical issue when sterility is essential (e.g., maintenance of cell cultures). Similarly, the surfaces of all materials and containers placed into the cabinet should be wiped with 70% ETOH to reduce the introduction of contaminants to the cabinet environment. This simple step will reduce introduction of mold spores and thereby minimize contamination of cultures. Further reduction of microbial load on materials to be placed or used in BSCs may be achieved by periodic decontamination of incubators and refrigerators. Material Placement Inside the BSC Plastic-backed absorbent toweling can be placed on the work surface (but not on the front or rear grille openings). This toweling facilitates routine cleanup and reduces splatter and aerosol formation during an overt spill. It then can be folded and placed in an autoclavable biohazard bag when work is completed. All materials should be placed as far back in the cabinet as practical, toward the rear edge of the work surface and away from the front grille of the cabinet. Similarly, aerosol-generating equipment (e.g., vortex mixers, tabletop centrifuges) should be placed toward the rear of the cabinet to take advantage of the air split described above. Active work should flow from the clean to contaminated area across the work surface. Bulky items such as biohazard bags, discard pipette trays and suction collection flasks should be placed to one side of the interior of the cabinet. Clean cultures (left) can be inoculated (center); contaminated pipettes can be discarded in the shallow pan and other contaminated materials can be placed in the biohazard bag right). This arrangement is reversed for left-handed persons.

Certain common practices interfere with the operation of the BSC. The autoclavable biohazard collection bag should not be taped to the outside of the cabinet. Upright pipette collection containers should not be used in BSCs nor placed on the floor outside the cabinet. The frequent inward/outward movement needed to place objects in these containers is disruptive to the integrity of the cabinet air barrier and can compromise both personnel and product protection. Only horizontal pipette discard trays containing an appropriate chemical disinfectant should be used within the cabinet.

66 Furthermore, potentially contaminated materials should not be brought out of the cabinet until they have been surface decontaminated. Alternatively, contaminated materials can be placed into a closable container for transfer to an incubator, autoclave or for other decontamination treatment. Operations Within A Class II BSC Many common procedures conducted in BSCs may create splatter or aerosols. Good microbiological techniques should always be used when working in a biological safety cabinet. For example, techniques to reduce splatter and aerosol generation will minimize the potential for personnel exposure to infectious materials manipulated within the cabinet. Class II cabinets are designed so that horizontally nebulized spores will be captured by the downward flowing cabinet air within fourteen inches of travel. Therefore, as a general rule of thumb, keeping clean materials at least one foot away from aerosol generating activities will minimize the potential for cross-contamination. The general workflow should be from “clean to contaminated (dirty)”. Materials and supplies should be placed in such a way as to limit the movement of “dirty” items over “clean” ones. Several measures can be taken to reduce the chance for cross-contamination when working in a BSC. Opened tubes or bottles should not be held in a vertical position. Investigators working with Petri dishes and tissue culture plates should hold the lid above the open sterile surface to minimize direct impaction of downward air. Bottle or tube caps should not be placed on the toweling. Items should be recapped or covered as soon as possible. Open flames are not required in the near microbe-free environment of a biological safety cabinet. On an open bench, flaming the neck of a culture vessel will create an upward air current that prevents microorganisms from falling into the tube or flask. An open flame in a BSC, however, creates turbulence that disrupts the pattern of air supplied to the work surface. When deemed absolutely necessary, touch-plate microburners equipped with a pilot light to provide a flame on demand may be used. Internal cabinet air disturbance and heat buildup will be minimized. The burner must be turned off when work is completed. Small electric “furnaces” are available for decontaminating bacteriological loops and needles and are preferable to an open flame inside the BSC. Disposable sterile loops can also be used. Aspirator bottles or suction fl asks should be connected to an overflow collection flask containing appropriate disinfectant, and to an in-line HEPA or equivalent filter. This combination will provide protection to the central building vacuum system or vacuum pump, as well as to the personnel who service this equipment. Inactivation of aspirated materials can be accomplished by placing sufficient chemical decontamination solution into the flask to kill the microorganisms as they are collected. Once inactivation occurs, liquid materials can be disposed of appropriately as non-infectious waste. Investigators must determine the appropriate method of decontaminating materials that will be removed from the BSC at the conclusion of the work. When chemical means are appropriate, suitable liquid disinfectant should be placed into the discard pan before work begins. Items should be introduced into the pan with minimum splatter, and allowed appropriate contact time as per manufacturer’s instructions. Contaminated items should be placed into a biohazard bag.

67 Decontamination Surface Decontamination - All containers and equipment should be surface decontaminated and removed from the cabinet when work is completed. At the end of the workday, the final surface decontamination of the cabinet should include a wipe-down of the work surface, the cabinet’s sides and back, and the interior of the glass. If necessary, the cabinet should also be monitored for radioactivity and decontaminated when necessary. Investigators should remove their gloves and gowns and wash their hands as the final step in safe microbiological practices. Small spills within the BSC can be handled immediately by removing the contaminated absorbent paper toweling and placing it into the biohazard bag. Any splatter onto items within the cabinet, as well as the cabinet interior, should be immediately wiped with a towel dampened with decontaminating solution. Gloves should be changed after the work surface is decontaminated and before placing clean absorbent toweling in the cabinet. Hands should be washed whenever gloves are changed or removed. Spills large enough to result in liquids flowing through the front or rear grilles require more extensive decontamination. All items within the cabinet should be surface decontaminated and removed. After ensuring that the drain valve is closed, decontaminating solution can be poured onto the work surface and through the grille(s) into the drain pan. Twenty to thirty minutes is generally considered an appropriate contact time for decontamination, but this varies with the disinfectant and the microbiological agent. Manufacturer’s directions should be followed. The spilled fluid and disinfectant solution on the work surface should be absorbed with paper towels and discarded into a biohazard bag. The drain pan should be emptied into a collection vessel containing disinfectant. A flexible tube should be attached to the drain valve and be of sufficient length to allow the open end to be submerged in the disinfectant within the collection vessel. This procedure serves to minimize aerosol generation. The drain pan should be flushed with water and the drain tube removed. Should the spilled liquid contain radioactive material, a similar procedure can be followed. Your supervisor or the Radiation Safety Officer should be contacted for specific instructions. Gas Decontamination - BSCs that have been used for work involving infectious materials must be decontaminated before HEPA filters are changed or internal repair work is done. Before a BSC is relocated, a risk assessment that considers the agents manipulated within the BSC must be done to determine the need for decontamination. The most common decontamination method uses formaldehyde gas.

Certification of Biological Safety Cabinets Containment Standards - The National Sanitation Foundation (NSF International) Standard No. 49 for Class II (Laminar Flow) Biohazard Cabinetry is the Standard for biological safety cabinets, and establishes performance criteria for biological safety cabinets and provides the minimum requirements that are accepted in the United States. This standard replaces the older NIH specifications that had previously been used. NSF Standard 49 incorporates specifications regarding design, materials and construction. Cabinets that meet the standard and were certified by the NSF bear an NSF 49 Seal. The operational integrity of a new BSC must be validated by certification before it is put into service or after a cabinet has been repaired or relocated. Relocating a BSC may break the HEPA filter seals or otherwise damage the filters or the cabinet. Each BSC must be tested and certified

68 at least annually to ensure continued proper operation. The Dean, Basic Biomedical Sciences will be responsible for scheduling certifications. Experienced, qualified personnel must perform on-site testing following the recommendations for field-testing (NSF Standard 49). Some basic information is included here to assist in understanding the frequency and kinds of tests to be performed. The importance of proper certification cannot be emphasized enough, since persons who manipulate infectious microorganisms are at increased risk of acquiring an occupational illness when their BSCs are functioning improperly. General Biosafety Issues

Biological Stains Fixatives and stains used for the preparation of tissues and cellular materials often have toxic properties (e.g. methylene blue and trypan blue are teratogens), requiring the use of impermeable gloves and appropriate ventilation. In addition several dyes used in conjunction with flow cytometry and visualization of nucleic acids are suspect carcinogens. Be sure the precautions you are taking are adequate. If in doubt, consult with your supervisor or Research Compliance and Safety. Incubators Incubators can become the inadvertent and undesired repositories of microorganisms. Although they may present a hazard to laboratory workers, most often they are a source of contamination of laboratory cultures. Besides the moist surfaces, rubber gaskets, the humidity trough (if present), and fan mechanism are areas in which contaminating microorganisms concentrate. It is recommended that an anti- microbial agent, such as Zepharin Chloride be added to the humidity source water; do not use sodium azide. In addition, the inner panels, trays, and the other removable parts should be autoclaved and the gaskets and non-removable parts wiped thoroughly with 70% ethanol every two months. Freezer and Liquid Nitrogen Storage Freezers containing potentially hazardous biological materials and toxins should be labeled accordingly. These freezers should be defrosted at least annually to prevent the accumulation of broken vials and excessive frost. Note that “frost-free” freezers allow small samples to thaw during warming cycles. Ethanol and other flammable solvents may not be placed in refrigerators or freezers that are not designed for flammable solvent storage. Moving the controls of a standard refrigerator or freezer to the outside is not acceptable, and does not allow for the storage of flammable solvents inside the altered unit. If you must refrigerate flammable solvents, use only a refrigerator or freezer meeting OSHA requirements for flammable solvent storage. Cells and virus stocks should be stored in sealed ampules and not in screw cap glass vials. Screw cap glass vials are permeable to liquid nitrogen (approximately 50% of the time) and therefore represent a source of contamination in the storage tank. Plastic screw cap ampules also leak and must be used with a heat-sealed sleeve to prevent contamination of the liquid nitrogen and

69 other samples. Upon thawing, screw cap vials may explode, producing an aerosol of glass and cell debris. If freezing manually, place ampules in the bottom of a beaker, cover with methanol and a dye, e.g., methylene blue, and transfer the entire beaker from refrigerator to freezer. The methanol provides even freezing and the dye will penetrate imperfectly sealed vials permitting their identification and elimination. When adding samples to liquid nitrogen storage repositories, be aware that the liquefied nitrogen may boil vigorously as warmer materials are added. Use only in a well-ventilated area. Liquefied nitrogen is a cryogenic gas and expands 700-fold upon vaporization; this may result in a rapid displacement of air. See the section on Laboratory Equipment Safety for addition safety equipment when working with cryogenic liquids. Ampoules to be thawed should be dropped into a plastic beaker containing 70% ethanol at 37C within a spongy bucket and covered immediately. Confirm the identification of the sample. Open the vial in a biological safety cabinet, by nicking the ampoule with a file near the neck. Wrap it in ethanol-wetted material and, holding the vial upright, snap the ampoule open at the nick. Add liquid slowly to dried material. Withdraw the suspension and mix in another vessel. Biological Spills and Decontamination

The following points should be followed in the event of a small, contained spill of biological materials or until assistance from your supervisor or Director, EHS is obtained: • If the substance is dry or nonvolatile, shut off hoods, close windows and doors, and vacate rooms. Label door with appropriate warning. Allow the aerosol to settle for at least 30 minutes before reentering room. • If the substance is volatile, leave on ventilation and vacate room, closing door. Label door with appropriate warning. Notify your laboratory supervisor and Director, EHS. • Assemble materials necessary for decontamination and don appropriate protective clothing, i.e., disposable lab coat, impermeable gloves. Note that surgical latex gloves are permeable to alcohol and that a respirator may be required if the substance is volatile. If you feel unsure of your ability to respond appropriately to the spill, or if you are not approved by supervisor for a respirator, do not attempt to clean up until your supervisor or IBC chair arrives. • For a liquid biological spill: pour the appropriate decontaminating solution (see below) on the spill, working from the perimeter inward. Decontamination Keeping biological waste separate from other waste streams is essential for any management program. Ideally, biological waste should be treated and destroyed on-site. Disposal of biological (medical) waste is becoming increasingly more regulated and costly. If unsure of the proper disposal route, contact the Research Operations Manager and the Environmental Health and Safety Officer before disposing of biological waste. All culture materials and biological specimens, including that from “normal” cultures and primary tissue, should be collected inside the biological safety cabinet. These materials should be autoclaved or otherwise chemically inactivated on at least a daily schedule.

70 Do not leave untreated waste in a corridor or public area. Hypochlorites or any other strong oxidizing material must not be autoclaved with organic material such as paper, cloth, oils, or volatile solvents This may produce toxic vapors or an explosion. Therefore, do not autoclave waste that has been treated with chlorine bleach. Do not autoclave materials contaminated with radioisotopes or toxic chemicals. These materials may volatilize and contaminate the autoclave and expose workers. The biological safety cabinet should be wiped down with an appropriate disinfectant (see below) prior to and at the end of each session. Disinfectants Alcohol - Isopropyl and ethyl alcohols in 70-90% concentrations may be germicidal against lipid- containing agents but are not effective against spores and infectious DNA. Note that 100% ethanol is not a good disinfectant. The major advantage of alcohols is that they are fast acting, evaporate rapidly, and leave no residue. Moreover, they can be combined with other disinfectants (quaternaries, phenolics, and iodine) to form tinctures further enhancing cidal action. Chlorine - A very active disinfectant, chlorine is cidal against a wide variety of gram-negative and gram-positive bacteria, bacterial spores and most viruses. Disinfect media with a 10% solution of chlorine bleach (5.25% hypochlorite or 52,500 ppm) for 15 to 30 minutes. Note that solutions deteriorate with age and are rapidly neutralized by organic matter. Its effectiveness may be enhanced by the addition of 0.1% solution of an ionic detergent. If used directly on a stainless steel surface, rinse thoroughly with water to prevent tarnishing and decomposition. Do not autoclave chlorine solutions. Iodophor - Characteristics of chlorine and iodine are similar. Iodophors are effective against gram-positive and gram-negative organisms, mycobacteria, and some viruses, and are most effective in acid solutions. Organic matter reduces effectiveness, but iodophors are less affected than hypochlorites. Do not autoclave since iodophors vaporize at 120 oF. Iodophors are stable in storage if kept cool and tightly covered. Regulated Medical/Bio-Hazardous Waste Management

What is biohazardous waste? Federal (EPA RCRA 40 CFR section 261.33), State (SD Legislative Rules, Article 74:35 Medical Waste) and local environmental laws (Revised Ordinances City of Sioux Falls, SD, Ordinance No. 41-02, Chapter 18 Garbage and Trash, Article V. Solid Waste, Regulated Medical Waste, Transfer and Recycling Facilities) consider biohazardous waste to be certain laboratory and medical treatment apparatus. The table below summarizes the disposal route for each of the biohazardous waste streams. Sharps • Syringes with or without needles • Broken glass, contaminated by biological or chemical waste • Scalpels, razors and lancets • Glass pipettes • Specimen tubes, slides,

71 Where to put for Container for Disposal Final Disposition disposal Place closed red sharps Sharps Red sharps container Autoclaving / containers in red with biohazard sign Disposal Company biosafety bag Glass disposal box - Broken Glass (not close and tape shut Picked up in lab Disposal contaminated) before pickup After 30 minutes, may Human or animal Decontaminate with be decanted down the blood, blood- Bleach to a final drain and flush with Sanitary Sewer products, body fluids concentration of 10% copious amount of water In 28 or 32 gallon Red Human Tissue Red Biohazard Bag Reusable Biohazard Autoclaving container Corrugated box marked Pathology Wastes Red biohazard bag Incineration pathology waste Cultures, infectious In 28 or 32 gallon Red agents and Red Biohazard bag Reusable Biohazard Autoclaving associated container biologicals Place in a red Place in biohazard Animal Carcasses Incineration biohazard bag freezer Any medical equipment or In 28 or 32 gallon Red Place in red biohazard disposables that Reusable Biohazard Autoclaving bag have the appearance container of medical waste Chemotherapy In a brown corrugated Waste Place in yellow box marked Incineration (including animal chemotherapy bag “Chemotherapy” bedding)

Human or animal blood, blood-products, body fluids and tissues Cultures, infectious agents and associated biologicals • Used Petri plates containing culture agar • Specimens from bottles, medical, pathology and research laboratories • Discarded live and attenuated vaccines • Wastes from the production of biologicals • Culture flasks Other laboratory wastes including but not limited to • Surgical drapes and absorbents • Protective gloves, disposable lab coats, or masks • Specimen containers

72 • All microorganisms constructed using rDNA Any medical equipment or disposables that have the appearance of medical wastes! • Agarose gels • PAGE gels • Sample buffers (tubes containing small amounts of liquid) • Membranes • Any disposable tubes, plates, dishes, cuvettes, including centrifuge, micro-centrifuge and conical tubes • Tips ( of any kind) Summary of Biosafety Levels

From CDC/NIH publication Biosafety in the Microbiological and Biomedical Laboratory , 4th Edition. Biosafety Level 1 (BSL1) Biosafety Level 1 is suitable for work involving well-characterized agents not known to cause consistently disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. The laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is neither required nor generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science. Biosafety Level 2 (BSL2) Biosafety Level 2 is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs from BSL-1 in that (1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists; (2) access to the laboratory is limited when work is being conducted; (3) extreme precautions are taken with contaminated sharp items; and (4) certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment. Biosafety Level 3 (BSL3) Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents that may cause serious or potentially lethal disease because of exposure by the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents. All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features. Note there are no USD laboratories meet the design requirements for working with BSL3 Agents at this time. Biosafety Level 4 (BSL4) Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease. Agents

73 with a close or identical antigenic relationship to Biosafety Level 4 agents are handled at this level until sufficient data are obtained either to confirm continued work at this level, or to work with them at a lower level. NOTE : There are no USD facilities suitable for working with BSL4 agents; thus, work with such agents at USD is prohibited.

Table 1. Summary of Recommended Biosafety Levels for Infectious Agents Biosafety Agents Practices Safety Equipment (Primary Facilities (Secondary Level Barriers) Barriers) 1 Not known to Standard Microbiological None required Open bench top sink consistently cause Practices Required disease in healthy adults 2 Associated with human BSL-1 practice plus: Primary barriers = Class I or BSL-1 plus: disease, hazard = II Biological Safety Cabinets Limited access Autoclave available percutaneous injury, (BSCs) or other physical ingestion, mucous Biohazard warning signs containment devices used membrane exposure for all manipulations of "Sharps" precautions agents that cause splashes Biosafety Policy defining or aerosols of infectious any needed waste materials; decontamination or PPEs: laboratory coats; medical surveillance gloves; face protection as policies needed 3 Indigenous or exotic BSL-2 practice plus: Primary barriers = BSL-2 facility plus: agents with potential Controlled access Class I or II BCSs Physical separation for aerosol from access corridors transmission; disease Decontamination of all or other physical may have serious or waste Self-closing, double- containment devices used lethal consequences door access Decontamination of lab for all open manipulation clothing before laundering of agents; Exhausted air not recirculated Baseline serum PPEs: protective lab clothing; gloves; Negative airflow into respiratory protection as laboratory needed 4 Dangerous/exotic BSL-3 practices plus: Primary Barriers = All BSL-3 plus: agents which pose high procedures conducted in Clothing change before Separate building or risk of life-threatening Class III BSCs or Class I or II entering isolated zone disease, aerosol- BSCs in combination transmitted lab Shower on exit with full-body, Dedicated supply and infections; or related air-supplied, exhaust, vacuum, and All material agents with unknown positive pressure decon systems risk of transmission decontaminated on exit personnel suit from facility Other requirements outlined in the text

74 Biosafety Levels for Vertebrate Animal Table 2 . Summary of Recommended Biosafety Levels for Activities in Which Experimentally or Naturally Infected Vertebrate Animals Are Used Biosafety Agents Practices Safety Equipment (Primary Facilities (Secondary Level Barriers) Barriers) 1 Not known to cause Standard animal care and As required for normal care Standard animal consistently disease in management practices, of each species. facility healthy human adults. including appropriate No recirculation of medical surveillance exhaust air programs Directional air flow recommended Handwashing sink recommended 2 Associated with human ABSL-1 practices plus: ABSL-1 equipment plus ABSL-1 facility plus: disease. primary barriers: Limited access Autoclave available containment equipment Hazard: percutaneous Biohazard warning signs appropriate for animal Handwashing sink exposure, ingestion, species; available in the animal mucous membrane Sharps precautions room. exposure. PPE: laboratory coats, Biosafety Policy gloves, face and respiratory Mechanical cage Decontamination of all protection as needed. washer used infectious wastes and of animal cages prior to washing 3 Indigenous or exotic ABSL-2 practices plus: ABSL-2 equipment plus: ABSL-2 facility plus: agents with potential Controlled access Containment equipment Physical separation for aerosol for housing animals and from access corridors transmission; disease Decontamination of cage dumping activities may have serious clothing before laundering Self-closing, double- health effects. Class I or II BSCs available door access Cages decontaminated for manipulative before bedding removed Sealed penetrations procedures Disinfectant foot bath as Sealed windows (inoculation, necropsy) that needed may create infectious Autoclave available in aerosols. facility PPEs: appropriate respiratory protection 4 Dangerous/exotic ABSL-3 practices plus: ABSL-3 equipment plus: ABSL-3 facility plus: agents that pose high Entrance through change Maximum containment Separate building or risk of life threatening room where personal equipment (i.e., Class III isolated zone disease; aerosol clothing is removed and BSC or partial containment transmission, or related Dedicated supply and laboratory clothing is put equipment in combination agents with unknown exhaust, vacuum and on; shower on exiting with full body, air-supplied risk of transmission. decontamination positive-pressure All wastes are systems personnel suit) used for all decontaminated before procedures and activities Other requirements removal from the facility outlined in the text

75 Summary of Agents

This list is not all-inclusive; contact the Chair, IBC for assistance determining the Biosafety Level for agents not on this list. Note that working with high titers or large volumes (e.g., > 1 liter) of these agents require higher levels of BSL facilities and procedures. From the CDC/NIH publication Biosafety in the Microbiological and Biomedical Laboratory , 4th Edition; more detailed summaries of these agents as well as others that are not listed, are available there. Additionally, the American Biological Safety Association (ABSA) has a web page (http://www.absa.org/riskgroups/index.html ) which lists Risk Group Classification for Infectious Agents. The listing includes classification by countries other than the US. BSL2 Bacterial Agents Bacillus anthracis Bordetella pertussis Brucella (B. abortus, B. canis, B. melitensis, B. suis) Burkholderia pseudomallei (Pseudomonas pseudomallei) Campylobacter (C. jejuni/C. coli, C. fetus subsp. fetus) Chlamydia psittaci, C. pneumoniae, C. trachomatis Clostridium botulinum Clostridium tetani Corynebacterium diphtheriae Escherichia coli (Cytotoxin-producing (VTEC/SLT) organisms) Francisella tularensis Helicobacter pylori Listeria monocytogenes Legionella pneumophila; other Legionella-like agents Mycobacterium leprae Mycobacterium tuberculosis, M. bovis Neisseria gonorrhoeae Neisseria meningitidis Salmonella - all serotypes including typhi Shigella spp. Treponema pallidum Vibrionic enteritis (Vibrio cholerae, V. para-haemolyticus) Yersinia pestis BSL2 Viral Agents (other than arboviruses) Hantaviruses – Hantaan, Puumala, Seoul, and Sin Nombre (plus others such as s El Moro Canyon virus), BSL2 facilities using BSL3 practices Hepatitis A Virus, Hepatitis E Virus Herpesvirus simiae (Cercopithecine herpesvirus [CHV-1], B-virus); large volumes are BSL4 Human Herpes viruses Influenza Poliovirus Poxviruses (smallpox, vaccinia, yaba, tanapox, monkeypox, plus others) Rabies Virus Retroviruses, including Human and Simian Immunodeficiency Viruses (HIV and SIV) Vesicular Stomatitis Virus

76 Radiation Safety USD has a radiation safety program under the authority of the USD Radiation Safety Officer. This program assures compliance with the Nuclear Regulatory Commission license to use radioactive materials. The USD Radiation Safety Office provides a range of radiation protection services, including training of laboratory personnel, inventory of all radioisotopes used on campus, receipt and delivery of all radioactive material and waste pickup and disposal. Individual Health and Safety Plan The Biological and Chemical Safety Policy provides a general outline of laboratory policies and procedures. This policy should be adapted by each research team (Principal Investigator, research staff and graduate students) to meet the specific needs in the laboratory by adding safety and health policies and procedures beyond those in this policy. The following is suggested list of information to be included: − Hazardous Materials being used in the laboratory − Required Training − Medical Monitoring − Registrations/Notifications/Permits − List of Laboratory Personnel − Special Emergency Procedures − Individual Laboratory Procedures − Departmental Policies and Procedures References • Fire Protection Guide on Hazardous Materials. National Fire Protection Association, • Quincy, MA (latest edition). • Laboratory Safety: Principles and Practices. Fleming, D. O. et al. American Society for • Microbiology. Washington, D.C. (latest edition) • Chemical Hazards of the Workplace. Proctor, N. and J. Hughes. J.B. Lippincott Co., • Philadelphia, PA (latest edition). • Prudent Practices for Handling Hazardous Chemicals in Laboratories. National Research • Council Committee on Hazardous Substances in the Laboratory. National Academy Press, • Washington, D.C. (latest edition). • Dangerous Properties of Industrial Materials. Sax, N. Irving. Van Nostrand Reinhold, New • York, NY (latest edition). • CRC Handbook of Laboratory Safety, Steere, N. ed. CRC Press, Inc., Boca Raton, FLA • (latest edition). • The Merck Index. Windholz, M., ed. Merck and Co. Inc., Rahway, N.J. (latest edition).

77 Appendix A. Example Glove Compatibility Chart

78 Appendix B. NIOSH Guide on Chemical Storage

(extracted from NIOSH School Chemistry Laboratory Safety Guide DHHS (NIOSH) Publication No. 2007–107 October 2006)

How Should Chemicals Be Stored?

Criteria for Storage Area • Store chemicals inside a closeable cabinet or on a sturdy shelf with a front-edge lip to prevent accidents and chemical spills; a .-inch front edge lip is recommended. • Secure shelving to the wall or floor. • Ensure that all storage areas have doors with locks. • Keep chemical storage areas off limits to all students. • Ventilate storage areas adequately. Organization • Organize chemicals first by COMPATIBILITY - not alphabetic succession (see Suggested Shelf Storage Pattern, below). • Store alphabetically within compatible groups. Chemical Segregation • Store acids in a dedicated acid cabinet. Nitric acid should be stored alone unless the cabinet provides a separate compartment for nitric acid storage. • Store highly toxic chemicals in a dedicated, lockable poison cabinet that has been labeled with a highly visible sign. • Store volatile and odoriferous chemicals in a ventilated cabinet. • Store flammables in an approved flammable liquid storage cabinet (see Suggested Shelf Storage Pattern, below). • Store water sensitive chemicals in a water-tight cabinet in a cool and dry location segregated from all other chemicals in the laboratory. Storage Don’ts • Do not place heavy materials, liquid chemicals, and large containers on high shelves. • Do not store chemicals on tops of cabinets. • Do not store chemicals on the floor, even temporarily. • Do not store items on bench tops and in laboratory chemical hoods, except when in use. • Do not store chemicals on shelves above eye level. • Do not store chemicals with food and drink. • Do not store chemicals in personal staff refrigerators, even temporarily. • Do not expose stored chemicals to direct heat or sunlight, or highly variable temperatures. Proper Use of Chemical Storage Containers • Never use food containers for chemical storage. • Make sure all containers are properly closed. • After each use, carefully wipe down the outside of the container with a paper towel before returning it to the storage area. Properly dispose of the paper towel after use.

79 Suggested Shelf Storage Pattern

A suggested arrangement of compatible chemical families on shelves in a chemical storage room, suggested by the Flinn Chemical Catalog/Reference Manual, is depicted on the following pages. − First sort chemicals into organic and inorganic classes. − Next, separate into the following compatible families.

Inorganics Organics 1. Metals, Hydrides 1. Acids, Anhydrides, Peracids 2. Halides, Halogens, Phosphates, 2. Alcohols, Amides, Amines, Glycols, Sulfates, Sulfites, Thiosulfates Imides, Imines 3. Amides, Azides*, Nitrates* (except 3. Aldehydes, Esters, Hydrocarbons Ammonium nitrate), Nitrites*, Nitric 4. Ethers*, Ethylene oxide, Halogenated acid hydrocarbons, Ketenes, Ketones 4. Carbon, Carbonates, Hydroxides, 5. Epoxy compounds, Isocyanates Oxides, Silicates 6. Azides*, Hydroperoxides, Peroxides 5. Carbides, Nitrides, Phosphides, Selenides, Sulfides 7. Nitriles, Polysulfides, Sulfides, Sulfoxides 6. Chlorates, Chlorites, Hydrogen Peroxide*, Hypochlorites, 8. Cresols, Phenols Perchlorates*, Perchloric acid*, Peroxides 7. Arsenates, Cyanates, Cyanides 8. Borates, Chromates, Manganates, Permanganates 9. Acids (except Nitric acid) 10. Arsenic, Phosphorous*, Phosphorous Pentoxide*, Sulfur *Chemicals deserving special attention because of their potential instability

80 Suggested Shelf Storage Pattern for Inorganics

Inorganic #10 Inorganic #7 ACID STORAGE Arsenic, Phosphorous, Arsenates, Cyanates, CABINET Phosphorous Pentoxide, Sulfur Cyanides ACID INORGANIC #9 STORE AWAY FROM Acids, EXCEPT Nitric WATER acid – Store Nitric acid away from other Inorganic #2 Inorganic #5 acids unless the Halides, Halogens, Phosphates, Carbides, Nitrides, cabinet provides a Sulfates, Sulfites, Thiosulfates Phosphides, Selenides, separate Sulfides compartment for nitric acid storage Inorganic #3 Inorganic #8 Amides, Azides, Nitrates, Nitrites Borates, Chromates, Manganates,Permanganates EXCEPT Ammonium nitrate - STORE AMMONIUM NITRATE AWAY FROM ALL OTHER SUBSTANCES

Do not store Inorganic #1 Inorganic #6 chemicals on the floor Hydrides, Metals Chlorates, Chlorites, Hypochlorites,Hydrogen STORE AWAY FROM WATER. Peroxide, Perchlorates, STORE ANY FLAMMABLE Perchloric acid, Peroxides SOLIDS IN DEDICATED CABINET

Inorganic #4 Miscellaneous Carbon, Carbonates, Hydroxides, Oxides, Silicates

81 Suggested Shelf Storage Pattern for Organics

Organic #2 Organic #8 POISON STORAGE Alcohols, Amides, Amines, Cresols, Phenol Imides, Imines, Glycols CABINET STORE FLAMMABLES IN A Toxic substances DEDICATED CABINET

Organic #3 Organic #6 FLAMMABLE Aldehydes, esters, Azides, Hydroperoxides, hydrocarbons Peroxides STORAGE CABINET STORE FLAMMABLES IN A FLAMMABLE ORGANIC #2 DEDICATED CABINET Alcohols, Glycols, etc. Organic #4 Organic #1 Ethers, Ethylene oxide, Acids, Anhydrides, Peracids FLAMMABLE Halogenated Hydrocarbons, ORGANIC #3 STORE CERTAIN ORGANIC Ketenes, Ketones ACIDS IN ACID CABINET Hydrocarbons, Esters, STORE FLAMMABLES IN A etc. DEDICATED CABINET FLAMMABLE Organic #5 Miscellaneous ORGANIC #4 Epoxy compounds, Isocyanates

Organic #7 Miscellaneous Do not store Nitriles, Polysulfides, Sulfides, chemicals on the floor Sulfoxides, etc.

82 Appendix C. Biological Safety Self-Audit Form

Building ______Room Number ______Principal Investigator ______

Date ______Audit Performed by______

Laboratory Personnel Present______Perform this self-audit every year, and with every new employee or student. Please consult your principal investigator or Unit Safety Facilitator for specific guidance, questions, or clarification.

A “No” answer requires corrective action or an explanation as to why the practice is not followed.

Description of Condition Yes/No Comments Standard Practices Eating and drinking, handling contact lenses, applying cosmetics, and mouth pipetting are prohibited in the laboratory or animal rooms. Store and consume food and drink outside of the designated area. Avoid contact with mucous membranes of the mouth, nose and eyes, as this is a route of transmission for many infectious agents. Use mechanical devices when pipetting all samples. Hands are washed after handling potentially infectious material, removing gloves, and before leaving the laboratory. Personal and environmental contamination can be effectively controlled with routine handwashing with regular soap and water. Access to the laboratory or animal room is restricted when infectious agents are manipulated. The director or principal investigator of a work area can restrict access. Personnel entering the area are made aware of potential hazards and symptoms of exposure. The type of infectious agent and the work environment will dictate the appropriate immunizations and participation of an employee in an occupations health program. Work surfaces are decontaminated at least once per day and always after a spill of potentially infectious material. Use fresh solutions of an effective disinfectant (e.g., 1:10 dilution of bleach, an iodophor such as Wescodyne, etc.) ensuring adequate contact time with the contaminated surfaces. Description of Conditions Yes/No Comments/Explanations Sharps are handled safely and alternatives are considered. Exercise caution when handling any contaminated sharps (e.g., hypodermic syringes and needles, broken glassware, etc.). Substitute plasticware for glass items whenever possible. Use hypodermic syringes and needles (preferably needle locking syringes) only when no other alternative is available. Evaluate the use of safer syringe/needle units, such as those with retractable guards or those that re sheathes the needle. Avoid recapping needles. However, if recapping is necessary, a written protocol that uses accepted procedures is required.

Safety Equipment Laboratory coats, gowns, or uniforms are worn in the

83 laboratory or animal room. Select appropriate personal protective equipment to prevent contamination of the employee’s work or street clothes. Remove this protective clothing before leaving the laboratory or animal handling area. Protective garments should be professionally laundered and not taken home by personnel. Closed shoes and long pants are recommended when working with infectious materials or hazardous chemicals. Gloves are worn when you anticipate contact with potentially infectious agents or contaminated surfaces. Wear suitable gloves (e.g., latex, nitrile, vinyl) when handling potentially infectious agents, or when contacting surfaces potentially contaminated with infectious materials. Please note that a different glove type may be needed when handling chemicals. Discard gloves when they are torn or overtly contaminated, and do not wash or reuse disposable gloves. Remove gloves when you complete work with infectious materials and before you leave the work area. Wash your hands after removing gloves. A biological safety cabinet is used whenever there is a potential or creating aerosols or splashes of infectious materials, or when high concentrations or large volumes of infectious agents are used. Use a biological safety cabinet when there is the likely presence of an infectious agent transmissible by the aerosol route. A properly operating and certified class I or II biological safety cabinet is used when performing procedures that might cause splashing, spraying, or splattering (e.g., blending, sonicating). A class II cabinet will protect the operator as well as the integrity of the sample or product. Certify the biological safety cabinet annually or whenever a cabinet is moved. Locate cabinets away from traffic patterns, doors and vents. Train personnel in the proper use of the cabinet. Please note that a biological safety cabinet is not a chemical fume hood, nor is it a substitute for good aseptic techniques.

Description of Condition Yes/No Comments/Explanation An autoclave is available. It is good practice to autoclave all laboratory wastes to minimize the possible hazards for personnel handling potentially infectious waste. However, autoclaving is not considered a final treatment. See #12 below. Handwashing facilities are provided . A sink is available for handwashing within the laboratory or animal room. Soap dispensers and appropriate toweling or hand dryers are available. Eyewash and emergency showers are readily accessible. This safety equipment is available for accidental spills or splashes of hazardous chemicals or biological materials, and employees have been trained in their use.

Special Practices Infectious material-specific training is provided. Before work commences, employees receive specific information on the infectious agent(s), including details on potential hazards, necessary

84 precautions to minimize exposure, and symptoms of exposure. Additional training is provided as necessary to reflect changes in procedure or new information. Training is documented and updated. Training for potential exposure to bloodborne pathogens. All employees working with human blood, tissues, or other potentially infectious material, receive annual OSHA training. The laboratory or animal room is appropriately posted and labeled. Label the laboratory or animal room entrance with the biohazard symbol and other information including the biosafety level, agent(s) in use, personnel contact information, and personal protective equipment required for entry or when handling infectious materials. Additionally, identify any equipment used for storage or manipulation of infectious agents (e.g., incubators, freezers, centrifuges, etc.) with the biohazard symbol. Hypodermic syringes and needles are controlled. Stocks of unused syringes and needles and those not in use are secured in locked drawers or cabinets. An inventory of purchases and distribution is kept. Laboratory personnel and animal handlers receive appropriate immunizations and/or enrolled in a suitable occupational health/monitoring program, when applicable. Personnel receive immunizations for the agents handled or potentially hazardous (e.g., rabies, hepatitis B), and possibly enrolled in a health monitoring program. Description of Condition Yes/No Comments/Explanation Standard operating practices and procedures are written and followed. Incorporate biosafety procedures in standard operating practices that address the hazards, manipulation, storage, and disposal of infectious agents. Review these practices at least annually or when new agents or procedures are adopted. Infectious substances are shipped appropriately. Personnel comply with federal and international regulations, and are trained to classify, identify, package, label, and document shipments of infectious substances for transport by air or ground. Accidents and spills of infectious material are addressed in an emergency plan or protocol. Personnel are familiar with protocols to handle spills or accidents involving infectious materials, including isolation and decontamination of the area equipment, and people. Potential exposures to infectious materials are reported. Report potential exposures to infectious material to the director of the laboratory or Animal facility, and the biological safety officer. Complete an accident report, and consult with safety officer for assistance, review incident and protocols to minimize or eliminate recurrence.

Waste cultures and stocks of infectious agents, and items in contact with infectious materials are disposed properly. Dispose of infectious materials (e.g., plastic culture plates, flasks that contain infectious agents), including mammalian cell culture wastes, and contaminated non-sharps items (e.g., gloves, paper) in a red biohazard bag, within a biohazard-labeled container. This container is covered when not in

85 use. See #12 below. Liquid infectious wastes (e.g., broth) are treated with a suitable disinfectant (e.g., 1:10 bleach) and discarded down the sanitary sewer. All hypodermic syringes and needles and other contaminated sharps items (e.g., razor blades, scalpels, Pasteur pipettes) are disposed properly. Collect sharps items in rigid plastic containers that are puncture resistant, leakproof, secured to preclude loss of contents, and labeled with the biohazard symbol. Discard these containers when they are ¾ full. See #12 below. Red biohazard bags and sharp containers are disposed properly.

Description of Condition Yes/No Comments/Explanation Fire extinguishers are checked annually.

Exits are marked.

Compressed gas bottles are secure.

Exit plan for emergencies is available and staff is knowledgeable of how to evacuate in an emergency.

Hood Certification occurs annually.

Access is limited or restricted. Current posted entry sign shows limited entry or hazards, with correct PPE identified. Other

Other

Other

86 Appendix D USD Laboratory Safety Inspection Form Building & Room #: ______Department: ______Date: Supervisor: Inspector: YES NO N/A Emergency Equipment Means of egress / exits clear and unobstructed Fire extinguishers in designated locations, accessible and unobstructed Safety Showers / Eyewashes labeled, accessible, free from obstructions, tested First aid kit available, maintained (i.e., no oral drugs) Personal protective equipment (PPE) available, appropriate to hazard and used Shoes, long pants, lab coats, glasses (i.e., protective clothing) worn when working in labs Spill kit available Emergency procedures posted Signs / Labels Emergency Contact List posted Refrigerators / freezers labeled “No Food & Drink” “No Flammables” or “Food & Drink Only” Biohazard / Radioactive Materials signs posted Other signs required (e.g., laser, magnets, bright light, etc.) Electrical Equipment Electric cords in good condition, no exposed wiring Ground Fault Circuit Interrupters (GFCI) in wet or high humidity areas Equipment properly grounded (e.g., ground plugs), 2-prong appliances not near sinks Extension cords for temporary use and cords / power strips not daisy chained Electrical panels free of obstructions Fume Hoods / Biological Safety Cabinets (BSC) / Laminar Flow Hoods Fume hoods, BSC, laminar flow hoods certified (dates: ) Hood sashes open / close properly and glass is intact Sash kept at 18” height while working and closed when not in use Hood kept running at all times Equipment used inside hood properly positioned / free of excess equipment Hood not used to store equipment, chemicals , nor waste Special purpose hoods (e.g., perchloric) Hazardous Material Chemical inventory available and up-to-date / updated annually Chemicals dated & initialed on receipt Chemical containers labeled with name of contents, capped and in good condition Corrosive chemicals stored below eye level Chemicals segregated by hazard (e.g., organics away from oxidizers, flammables from acids) Highly reactive chemicals stored in chemical-safe refrigerator Flammable solvents stored in approved safety cans or solvent storage cabinets No combustibles stored within 3 feet of the ceiling Secondary containers used to transport chemicals outside the lab Hazardous materials near sinks / drains inside secondary containers

87 Compressed Gas Storage Gas cylinders properly marked, stored / secured in upright position Stored cylinders tightly capped & kept to minimum Regulators, connections and tubing in good condition, valves closed with not in use Complies with NFPA (i.e., max of 3 flammable, oxygen / health hazard cylinders per 500 sq ft) For toxic gases, leak sensors / alarms in place, regularly checked and calibrated Training / Documentation / Publications YES NO N/A Laboratory workers trained / instructed in potential hazards and lab safety practices Lab specific training (SOPs, chemical segregation, equipment handling, etc.) documented Copy of all safety manuals (Radiation, Chemical, biological) available MSDSs available, updated, accessible (CD or hard copy) Laboratory workers received chemical / waste training All personnel in lab completed annual refresher training Hazardous Waste Management Waste containers labeled and chemical compositions identified Broken glassware, sharps, pipette tips segregated and properly disposed Waste stored in secondary containment Chemical waste stored in a “designated” area Biohazard containers properly used where needed (e.g., autoclave bags, sharps containers) Biological Safety Issues Biological and/or infectious agents in use (RG ) Biological use authorizations on file and current Laboratory furniture appropriate and easily decontaminated Biological waste bags with biohazard symbol in hard-sided, closed container Autoclave use log available / current and autoclaves tested after 40 hours of combined use Bloodborne pathogen program (workers trained & offered vaccinations - documented) Radiation Safety Issues NRC form 3 posted and Caution Radioactive Materials signs / labels used Absorbent paper used on work surfaces Radiation survey meter available and calibrated (date: ) Radioisotope inventories available and current Radiation survey performed monthly and contamination cleaned Radiation dosimeters used, if appropriate Radioactive waste segregated by isotope, labeled, activity recorded Radiation survey of work area performed at conclusion of procedure Lasers, UV, RF sources (Laser Class ) Vacuum Equipment Glass Dewars wrapped / shielded Vacuum pump belt guards in place Protective shatterproof shields used when vacuum equipment used.

Comments:

Discussed results with lab PI / supervisor Date deficiencies corrected / resurvey

88