2012 Annual Biosafety & Chemical

Safety Training

Topics

• Emergency Response in Laboratories

• Chemical /General Lab Safety

• Biosafety and Bloodborne Pathogens

• Working with the IBC

Emergency Response in Laboratories

Lab Accidents Happen

Chemical splash to eyes Chemical burns to skin Thermal burns Infectious exposures Needle punctures Lacerations Trip/slips/falls

Chemical Splash to Eyes Immediately flush with water for at

least 15 minutes (remove contacts)

Report to the Emergency Room or call 3333

Keep all eye wash stations free of obstructions

Chemical Exposure to Skin Immediately drench with water for at

least 15 minutes

Remove affected clothing

Report to Emergency Room or call 3333

Always keep shower stations free of obstructions

General Chemical Spill Procedure

S top & evaluate size & severity of spillP rotect any coworkers at risk I solate the spill by closing doorsL aunch response action (call x4220)L ocate MSDS

Remember….. S. P. I . L . L

General Chemical Spill ProcedureCALL x4220Large Spills (greater than 1 liter)Any spilled quantity of

Highly Hazardous MaterialsAcutely Toxic Materials

Any spill you cannot clean up yourselfReport all incidents to:

PI/Lab Supervisor AO/R&D; R&D Safety Officer Employee Health/ER if injured

Natural Gas Leak EmergenciesIf you smell or suspect it…1. If minor, try to shut off valve(s).2. If strong or cannot shut off valve(s), cease all

operations immediately and DO NOT operate electrical devices (DO NOT unplug/ plug or turn on/ off anything)

3. Evacuate the area (DO NOT use elevators)4. From a safe location, contact Police Service at (305)

575-3333

Chemical /General Lab Safety

OSHA’s Lab StandardChemical Hygiene Plan: Provides policies and procedures for working safely in labs Outlines the proper use of engineering controls (e.g. fume

hoods) and personal protective equipment Lab-specific SOPs to minimize chemical exposures

The Chemical Hygiene Plan is in each laboratory

The Miami VAHS Chemical Hygiene Officer is the R&D Safety Officer.

Material Safety Data Sheets Allow employees to better understand how to safely work

with hazardous chemicals.

Contains key information for a specific chemical, including health hazard information and control measures such as proper personal protective equipment, physical hazards and properties, handling and storage requirements, and emergency procedures.

All lab personnel should know how to read a MSDS and should review the MSDS’s for chemicals used or stored in their work area. This will help prevent accidents and increase safety awareness.

Material Safety Data SheetsInformation includes:

Chemical Name/ Synonym Manufacturer/Distributor Ingredients Chemical Characteristics Flammability/Reactivity Exposure Limit /Controls Health Hazard Info

Signs & Symptoms of Exposure

Disposal Guidelines

Material Safety Data Sheets

Each lab must maintain its own chemical inventory, and MSDSs (paper). You can access electronically

via manufacturer’s website or viahttp://vaww.ceosh.med.va.gov/ceosh/MSDS.shtml

Chemical InventoryPer VA’s regulation, must be updated twice a year.

Health; Flammable; Reactive;Contact; Radioactive; None

All acronyms must be defined

CHEMICAL INVENTORY - VA Healthcare System, Miami FL

Service: Research Inventory Date:

Room No(s): Investigator(s)/Supervisor(s):

UM in Cntnr

UMNo.

CntrsManufacturer Specific Hazard(s) YES NO

100 mg 2 Sigma-Aldrich; Tocris Health√

500 mL 1 Sigma-Aldrich Health; Contact √1 G 1 Sigma-Aldrich Health; Flammable √1 L 1 Fisher Scientific Health; Reactive; Contact √

MSDS ON FILE

Full Chemical Name

8-Cyclopentyl-1,3-dipropylxanthine or DPCPXAcetic acid volumetric standard, 1.0NMethanolSodium hydroxide solution, 10N

Basic Chemical Hazard Classesoxidizer

corrosive

flammable

gas

poison

Oxidizer Agents that react with reducible

material to initiate or promote combustion

Must not be stored with flammable solvents or other organic chemicals

Common oxidizers:

- Hydrogen peroxide (concentrated)

- Many nitrates and nitrites- Sulfuric, nitric, and chromic acids

Corrosives

Acids & Bases Tissue damage React violently with water and

each other Do not store acids & bases

together Separate oxidizing acids (nitric,

sulfuric) from organic acids (acetic acid, phenol)

Flammable Liquids

Liquid with a Flashpoint < 100oF Volatile, evaporate quickly, may lead to

explosive concentration in air Always store large quantities in rated

flammable storage cabinets Only keep small amounts out on

benchtops and shelves Do not store in refrigerators or freezers

not specifically designed for flammable chemical storage

GasFlammable gas (CO, H2)

Flashpoint < 140oF Ignites easily, burns rapidly

Non-flammable gas (including compressed gas) (N2, CO2) Purified gas in pressurized tank Tank rupture, toxic atmosphere,

oxygen displacement

Store upright, secure with a double chain to prevent falling

Explosive/Shock Sensitive

Thermodynamically unstable material

Explosion caused by shock or chemical reaction

Picric acid, 2,4-dinitro-phenol, organic azides

Store minimum quantities; discard before expiration date

Toxics/Poisons/Carcinogens Harmful or fatal if inhaled, ingested,

absorbed (Nicotine, Pesticides, Osmium Tetroxide)

Carcinogen refers to any substancethat is an agent directly involved in the promotion of cancer or in the increase of its propagation. (Dimethyl sulfate, Chloroform, Formaldehyde/ Formalin/Paraformaldehyde)

Chemical Exposure Pathways Inhalation – Chemical vapors or dust

Ingestion – Hand hygiene is important!

Injection – Needlesticks or broken glass

Absorption – Direct skin contact due to not wearing gloves, or wearing inappropriate gloves

Inhalation and absorption are most common

Chemical Fume Hoods

Primary control for inhalation protectionMust limit storage & clutter for proper

functioningKeep sash at or below recommended level Perform all work 6” inside the hoodKeep lab doors closed to ensure negative

room pressure to the corridor and proper air flow into the hood.

Chemical Resistant GlovesGlove selection is important:

– Variety of glove materials are available– No universal glove that is appropriate for

all chemicals– All chemicals eventually penetrate all

common glove materials– Gloves may appear to be intact even

when they are not

Chemical Resistant Gloves

Poor protection against many non-aqueous hazardous chemicals

Many people have allergies to latex Latex is a sensitizer, meaning that

repeated contact may cause you to develop an allergy

Latex powder can trigger allergic reactions in your fellow employees

Latex Gloves are strongly discouraged

Chemical Resistant GlovesNitrile gloves most commonly used in research labs

Pros:Low cost, good dexterity, resistance to wide range of chemicals (acids, bases, aliphatic solvents), resistant to abrasion and puncture

Cons:Poor protection against acetone, acetonitrile, tetrahydrofuran, halogens,phenol (seek alternative gloves, such as butyl rubber)

ONE GLOVE REMINDER If transporting any hazardous

materials outside the lab: HazMat = Glove

Common Surfaces = No Glove

Gloves should never touch door knobs, elevator buttons, etc.

Eye & Face ProtectionTypes of protection: Safety Glasses Safety Goggles – better protection Face Shield – for splash hazards Prescription glasses do not qualify!

Glass lenses, no side shields Contact Lenses are discouraged in labs

Can react to chemical exposure and can interfere with eye washing

No sandals or open-

toed shoes in the

laboratory

No food or beverages in the lab! Imminent health hazard OSHA regulation COMMON SENSE!

Throw food wrappers away before you enter the lab.

Chemical & Hazardous Material StorageKeep volumes to a minimum

Avoid ordering more than a few weeks’ supply at a time

Do not store chemicals on high shelves, under sinks, or anywhere else awkward to reach

Use carts or bottle carriers to transport bottles between labs

Flammable & Combustible Chemicals

Never order in bulk containersPrefer 1 gallon size limit.

Labs with 10+ gallons need flammable storage cabinet

Segregate Incompatibles Separate Acids and Caustics

Separate Organic and Mineral acids

Separate Oxidizers from all Organics

Keep flammables in rated cabinets and away from any ignition sources

No combustibles (cardboard, paper) in or on your flammable storage cabinets

Peroxide-Forming Chemicals Repeated exposure to air causes peroxide buildup

Unstable, potentially explosive

VERY dangerous, very expensive ($5-10k) to dispose

Monitor expiration dates, order in small quantitiesethyl etherpicric acid1,4-dioxaneisopropyl ether

Biosafety and Bloodborne Pathogens

Biosafety in the Laboratory• Primary goal of biosafety is to reduce or

eliminate exposures to potentially hazardous biological agents through the use of containment.

• Biosafety guidelines are designed to protect both laboratory personnel and the community and environment outside of the laboratory.

Risk Groups

Risk Group Agent Associated with Disease inHealthy Adults

Preventive or TherapeuticIntervention Available

RG1 NoRG2 Yes – Rarely serious disease Often AvailableRG3 Yes – May be serious or lethal disease May Be AvailableRG4 Yes – Likely serious or lethal disease Not Usually Available

Biological agents are categorized in Risk Groups (RG) based on their relative risk

Four RGs - RG1 to RG4

Biosafety Level (BSL)• Prescribe procedures and levels of containments for the

particular agent used • Four BSLs - BSL-1 to BSL-4 based on risk assessment

Biosafety Level Risk Group Examples

BSL-1

Individual Risk: LOW Community Risk: LOW

Escherichia coli, K12 based strains Baculovirus

BSL-2 Individual Risk: MODERATE Community Risk: LOW

Salmonella Staphylococcus Hepatitis B And C Viruses Adenoviruses

BSL-3 Individual Risk: HIGH Community Risk: MODERATE

Mycobacterium tuberculosis Yellow fever virus

BSL-4 Individual Risk: HIGH Community Risk: HIGH

Ebola virus Herpes B or Monkey B virus

BSL-1 containment is typically required for lab work involving:• Biological agents that meet the definition of RG1 (i.e. not associated

with disease in healthy adult humans);• Biological materials not suspected of containing RG2 or higher in a

quantity or form that may cause human disease (e.g., many soils and nonprimate animal cells);

• Biological agents or materials not characterized by the supplier as RG2 or higher;

• Transgenic or wild-type lab animals (e.g., rodents) that can be contained and are:– Free of zoonotic disease– Not infected with, implanted with, or containing RG2 or higher agents or materials

BSL-2 containment is typically required for lab work involving:• Biological agents/materials categorized as RG2 or may contain RG2

agents;• Bloodborne pathogen materials (e.g., human blood, tissues or cells)• Nonhuman primate tissues or cells;• Lab animals (e.g., rodents) infected with, implanted with, or containing

RG2 agents or materials (e.g., infected with a human pathogen or containing a xenotransplant of human cells)

• Tissues or cells potentially containing an RG2 agent

Three Components of Biosafety• Laboratory practices and techniques

– Primary goal is to minimize exposures to biological agents from inhalation, ingestion, or skin contact

– Standard microbiological techniques applied

• Safety equipment– Primary barriers act as a first line of defense against exposures to

hazardous agents (BSCs, PPE)

• Facility design– Secondary barriers protect laboratory workers inside the

laboratory and the community outside of the laboratory and varies depending on the biosafety level (limiting access, autoclaves for decontamination, handwashing facilities)

Biosafety Level 1 Practices• Access does not have to be restricted; however, doors cannot

be propped open• Handwashing after glove removal and prior to leaving

laboratory• Eating, drinking, smoking and applying cosmetics prohibited• Mouth pipetting prohibited• Sharps handled safely• Work surfaces decontaminated• Decontamination of biological waste

Biosafety Level 1: Safety Equipment• Laboratory coats• Gloves • Eyewear protection for splashes

and spills• Closed-toe shoes• Special containment equipment or

facility design is not required, but may be used as determined by a risk assessment.

Biosafety Level 2 Practices (BSL-1 Practices Plus):

• Personnel have specific training in handling pathogenic agents• Access to the laboratory is restricted when work is being

conducted.• Biohazard signs must be posted.• Laboratory personnel must be provided with medical

surveillance and offered appropriate immunization.

Biosafety Level 2: Safety Equipment• Biological safety cabinet (BSC) needed when aerosols and

splashes will be generated• Centrifuge safety cups• Face protection worn to minimize exposures to splashes

and aerosols outside BSC• Protective clothing worn only in laboratory• Gloves worn when handling infectious materials or

contaminated equipment• Decontamination procedures

Biological Safety Cabinets• Class II Type A2 is the most common type• Optimal performance:

grills free of clutter sash as low as possible no open flame allowed inside BSC

• Not designed to work with volatilechemicals

• Annual certification is mandatory

Biological Safety CabinetsBiological Safety CabinetsClean Dirty

UV lamps are not required or recommended in BSCs. If operated properly, BSCs do not need UV lights.

If installed UV lamps must be:• Cleaned weekly to remove dirt and dust

(they block germicidal effectiveness of UV light)• Checked periodically to ensure the appropriate intensity of

UV light is being emitted• Turned off when the room is occupied to protect eyes and

skin from UV exposure **can burn the cornea and cause skin cancer

Ultraviolet Lamps in BSCs

Ideal Vacuum Line Protection

Biohazardous Waste Containers

Sharps Precautions

• Never break, bend, resheath or reuse syringes or needles

• Always dispose of sharps in approved sharps containers

Bloodborne Pathogens (BBPs)Bloodborne Pathogens (BBPs)• OSHA Bloodborne Pathogens Standard (29 CFR 1910.1030)

• Standard is designed to protect workers from health hazards associated with occupational exposures to blood and other potentially infectious material (OPIM)

• HIV, HBV, HCV, and SIV

• Biological agents that cause: syphilis malaria viral hemorrhagic fever

Blood is a Potentially Infectious Material (PIM)

• OSHA’s definition of blood includes: Human and nonhuman primate blood

Human and nonhuman primate blood products

Human and nonhuman primate blood components such as plasma and platelets

Other Potentially Infectious Material (OPIM)

Human body fluids including: semen vaginal secretions breast milk cerebrospinal, pleural, synovial, pericardial, peritoneal, and

amniotic fluids any body fluid visibly contaminated with blood

Unfixed human and nonhuman primate tissues or organsHuman and nonhuman primate cells or cell lines

Other Potentially Infectious Material Is Not:

• Tears• Sweat• Saliva• Urine

Unless visibly contaminated with blood

OSHA BBP Standard

• Written Exposure Control Plan• Identification of all job titles with potential exposures to

PIM• Control of exposures to PIM• Hepatitis B vaccination offered• Communication of potential hazards to workers• Records maintained

Required Labeling• OSHA Standard requires that the biohazard symbol be

affixed to any equipment that comes into contact with biohazard agents: containers of regulated waste Refrigerators/freezers incubators containers used to ship, store, or transport

blood and OPIM

Bloodborne Pathogen SpillsBloodborne Pathogen Spills

Alert people in immediate area of spill and post signs to keep people out of the area to prevent spread of the contamination.

Wear the appropriate PPE to clean up spill: disposable gloves, lab coat, fluid resistant surgical mask, and safety glasses or goggles.

Pick up sharps with tongs/forceps and discard into sharps container. Cover spill with wipes or paper towel.Spray spill with a freshly prepared 1:10 dilution of bleach and allow

it to stand for at least 20 minutes.Wipe up the spill with paper towels, working from the edge toward

the center.• Wipe the area again with fresh paper towels soaked in disinfectant.• Place towels in a double-lined biological waste box.

Disinfection and DecontaminationMethods

Disinfection and DecontaminationMethods

• Alcohols (70 % isopropyl or ethyl alcohol solution) are effective for routine disinfection of surfaces

• Sodium hypochlorite (Bleach), 1:10 bleach dilution should be used for liquid biological waste decontamination

• Autoclaves

What If A BBP Exposure Occurs?• Immediately treat the exposure site

Needlesticks and cuts should be washed with soap and water, apply direct pressure if needed

Splashes to the nose, mouth, or skin require flushing with water or saline for 3-5 minutes

Splashes to eyes: remove contact lens immediately if worn, go to eyewash station and immediately rinse eye(s) and inner surface(s) of eyelid(s) with water continuously for 15 minutes

Go to the Emergency Room Notify your Supervisor Notify the Research Safety Officer and Research

Administration (Required by ORO)

Working with the IBC• NIH requires all labs working with recombinant DNA to

register with their local IBC• Recombinant DNA (rDNA)

– DNA constructed in vitro from separate DNA segments that can replicate and/or express a biologically active polynucleotide or polypeptide in vivo

– Synthetic DNA that has the potential of generating a hazardous product in vivo

UM’s IBC policy requires that any recombinant work, exempt or non-exempt, be registered with the IBC.(http://uresearch.miami.edu/default.asp?p=177)

Working with the IBCWhen filling out the IBC forms make sure you pay special attention to:1. What section of the NIH Guideline

applies to you2. Risk Groups and Biosafety Levels3. Be as specific as possible in regards to

your procedures, hosts, vectors, and genes

4. Always include the vector maps of your viral constructs

Call: 305-243-2311

Working with the IBC• When completing IBC forms always include the vector maps

Plasmid cloning vector in E. Coli Lentiviral vector Adeno-asssociated viral vector

Summary of NIH Guidelines• Section III-C: Experiments that require IBC, and Institutional Review Board

(IRB) approvals, and Recombinant DNA Advisory Committee (RAC) review before initiation.– Experiments involving the transfer of rDNA into human subjects.

• Section III-D: Experiments that require IBC approval before initiation. – Experiments involving the introduction of recombinant DNA into RG2, RG3 or RG4

agents for use in animal experiments. – Using adenovirus, adenovirus-luciferase or adeno-associated virus to transfect cells– Cloning a pathogen into a lower eukaryotic or prokarytoic cell– Insertion of KSHV (Kaposi’s sarcoma-associated herpesvirus) or RRV (Rhesus

Rhadinovirus) genes into defective lentiviral vectors– Creation of transgenic animals or knockout animals that leave genetic material in

the animal as part of the silencing of gene. (purchase or transfer of previously created transgenic rodents is exempt)

Summary of NIH Guidelines• Section III-E: Experiments that require IBC notice simultaneously with

initiation. – Experiments in which all components are derived from non-pathogenic

prokaryotes or non-pathogenic lower eukaryotes– rDNA with less than 2/3 of a eukaryotic viral genome (and no helper virus)

used exclusively in tissue culture (inserting DNA sequence that encode reporters that are measured (lacZ, luciferase, eGFP, etc))

• Section III-F: Experiments that are exempt from NIH Guidelines. However, registration with the UM IBC is required .– rDNA molecules not in organisms or viruses– rDNA that cannot replicate or express in vivo– rDNA molecules that consist entirely of DNA segments from a single

nonchromosomal or viral DNA source

More Questions...Reference materials (BMBL, NIH

Guidelines)

H&S Office x4220

Research Safety Officer x4436

Research Office x3179

Lab specific information should be obtained from lab supervisor or Principal Investigator

Things to Note

• No one should enter the lab areas without authorization. Need a VA Badge.• Visitors must be accompanied by a VA employee at all times. • Contractors must sign in with the Main Research Office prior to working.• All unknown personnel entering a research area must be challenged

– Example – lost patients, visitors without escorts– Can I help you?– What are you doing here?– Can’t access this area without authorization

• All lab door should be closed and locked at all times.– Closed for negative pressure– Locked for security – theft is a big issue/concern

• Computer must be locked when you are not at them.• Remove all copies from printers immediately regardless of whether you consider

it to be sensitive information