Duke Laboratory Safety Manual

Reviewed 04/2012

Laboratory Safety Manual

Introduction

FORWARD

Duke University is committed to providing a safe work environment for learning, teaching and
research. The Occupational and Environmental Safety Office (OESO) promotes workplace safety
through various policies and programs, many of which are outlined in this manual. The
following sections are meant to provide general laboratory safety procedures.

Each laboratory is a unique environment, and site-specific hazards must be addressed by each
supervisor/principal investigator when establishing proper work practices. It is each laboratory
professional’s duty to conduct their work in a responsible manner, and to take all necessary
precautions to protect themselves and others in the area from exposures to hazardous
materials. Upon request and during laboratory audits, OESO will assist in performing a risk
assessment for procedures of moderate to high risk, aid the lab in evaluating the risk, and
help with the development of written standard operating procedures (SOP).

If work practices currently being employed in your laboratory do not comply with those in this
manual, please make the necessary changes. An OESO representative can be reached for
questions by calling 919-684-8822 or 919-684-2794.
 Section 1 Biological Safety
Chapter 1
Introduction and Purpose

Laboratory Safety Manual

Section 1
Biological Safety

INTRODUCTION

PURPOSE
This section has been prepared to provide the laboratory personnel at Duke University with the
information necessary to protect them and the surrounding environment from hazards
associated with the use of biological materials. The guidelines which follow provide a means for
evaluating the risks of work involving biological materials and introduce the proper handling
practices which will minimize the risk of an occupational acquired infection. History has shown
that if not handled appropriately, infectious agents can be transmitted to laboratory
employees, and rarely, to people outside of the laboratory. Biohazardous materials are those
which are either known to cause, or that present a potential risk to the health of humans or
animals. Such materials would include, but are not limited to: bacteria, fungi, viruses, parasites,
rickettsia, rDNA toxins, human blood and unfixed human tissues. Work with biohazardous
material(s) requires consultation with the OESO – Biological Safety Division (919-684-8822).

Examples for Safe Handling of Biohazardous Materials

Material Precautions/ Employee Health Waste Disposal Shipping Training
Containment Services Requirements
Human- • BBP Exposure • Hepatitis B • All waste • Classify and • New
derived Control Plan (see vaccine decontaminated package Employee
(e. g. Appendices for this according to Duke according to I Orientation
blood, section) • All exposures Medical Waste ATA’s DGR
body reported to Management Policy. • General Lab
fluids, • BSL 2 (see BMBL* EOHW Exposure • Receipt or Safety (annual
tissue, or "Biosafety Hotline, 115 (on shipment of updates)
cells, Levels" of this campus) and 684- “select agents”
etc.) section) 8115 (off must comply • Shipping
campus) with 42 CFR Part training every 2
• BSL2 SOP 73. years
• Complete
the Report of • Importation of • BSL2 training
Work Related infectious
Injury or Illness substances • Bloodborne
form. requires a Pathogens
permit. training
 Section 1 Biological Safety
Chapter 1
Introduction and Purpose

Material Precautions/ Employee Waste Disposal Shipping Training

Containment Health Services Requirements
Known (or • Refer to BMBL* • Contact • All waste • Classify and New Employee
suspect) or "Biosafety EOHW at 684- decontaminated package Orientation
human or animal Levels" of this 3136 for according to Duke according to I
pathogens (e. g. section for vaccination Medical Waste ATA’s DGR • General Lab
HIV, appropriate BSL information. Management Safety (annual
Rickettsial or ABSL. Policy • Receipt or updates)
agents, • All exposures shipment of
Salmonella, etc.) • Refer to reported to “select agents” • Shipping
infectious agent EOHW Exposure must comply training every 2
MSDS** Hotline, 115 (on with 42 CFR years
campus) and Part 73.
•BSL2 SOP 684- 8115 (off • BSL2 training
campus) • Importation
of infectious
• Complete the substances
Report of Work requires a
Related Injury permit
or Illness form.
Recombinant • Follow NIH • Contact • All waste • Classify and • New
DNA/Transgenics Guidelines (also EOHW at 684- decontaminated package Employee
see 3136 for according to Duke according to I Orientation
"Recombinant vaccination Medical Waste ATA’s DGR
DNA" in this information. Management • General Lab
section) Policy. Safety (annual
• All exposures updates)
• Visit Duke's IBC to human
website to pathogens are • Shipping
complete reported to training every 2
appropriate EOHW Exposure years
registration Hotline, 115 (on
campus) and
• If material is a 684-8115 (off
biological vector, campus).
use appropriate Exposures to
BSL for wild type rDNA are
agent. See reported to
rDNA/viral vector OESO-Biological
webpage for Safety 684-
more information 8822.
and the rDNA
registration form • Complete the
for IBC Report of Work
registration Related Injury
information. or Illness form.
If BSL2 or above,
must have a SOP.
* Biosafety in Microbiological and Biomedical Laboratories, 5th ed.,CDC/ NIH, 2007: (http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm)
** Health Canada, Laboratory Center for Disease Control; Office of Biosafety: (http://www.phac-aspc.gc.ca/msds-ftss/index.html
 Section 1 Biological Safety 
Chapter 2  
Routes of Exposure 
 
ROUTES OF EXPOSURE 
 
There are four main routes of exposure that one must try to avoid when working with 
biohazardous agents in the laboratory. These would include percutaneous injuries, inhaling 
infectious aerosols, exposure to mucous membranes, and ingestion.  
 
Percutaneous injuries  
Percutaneous injuries can result from needlesticks, cuts or abrasions from contaminated items. 
These exposures are particularly serious because of the potential for immediate entry of the 
agent into a normally sterile bloodstream. All sharps items should be handled and disposed of 
as noted in the Waste Management section.  
 
Inhalation of aerosols  
Many laboratory procedures can cause the aerosolization of infectious agents. Some of these 
procedures include the use of vortexes, blenders and sonicators. Proper work practices must 
be implemented to minimize the aerosolization of all materials, especially those which are 
known to be transmitted by the aerosol route (e.g., Adenovirus, Vaccinia virus, Mycobacterium 
tuberculosis, etc.). See “Laboratory Equipment” for more information about minimizing and 
containing aerosols in the laboratory.  
 
Mucous membrane  
Exposure of mucous membranes to infectious agents can lead to occupationally acquired 
infections. Mucocutaneous exposures can result from splashes to the eyes, nose or mouth, or 
by inadvertent inoculation via contaminated hands. Face protection should always be used if 
there is a likelihood of splash or splatter.  
 
Ingestion  
Accidental ingestion of biohazardous materials can result from improper personal hygiene in 
the laboratory. Food and drink are prohibited in all areas of the laboratory in which work is 
conducted with potentially infectious materials. Hands must always be washed before leaving 
the laboratory, and immediately if visible contamination occurs.  
 
 
 Section 1 Biological Safety 
Chapter 3 
Standard Laboratory Practice and Technique 
 
STANDARD LABORATORY PRACTICE AND TECHNIQUE  
 
Personal Protective Equipment  
Once a biological hazard has been identified, the supervisor and employee must agree on 
the appropriate personal protective equipment (PPE) to be worn as the primary barrier of 
protection. PPE may include, but is not limited to face protection, lab coats and gowns, 
respirators, and shoe‐covers/booties. Supervisory personnel are responsible for the initial 
demonstration and periodic follow‐up of proper use. Appropriate PPE should be donned before 
handling potentially hazardous biological materials and removed immediately and replaced if 
gross contamination of the equipment occurs. PPE should be removed before exiting the 
laboratory.  
 
Face Protection: When splash or splatter of infectious substances or other biological 
materials is anticipated, appropriate face protection should be worn if work is 
performed outside a biological safety cabinet. Such equipment would include but is not 
limited to goggles, side‐shielded safety glasses and chin length face shields.  
 
Lab Coats and Gowns: Long sleeved lab coats or gowns should be worn to protect skin 
and street clothes from contamination. In circumstances when splash or splatter is 
anticipated, the garment must be resistant to liquid penetration. A cuffed lab coat or 
gown should be worn when working with potentially infectious materials, and MUST be 
worn when working with agents requiring Biosafety Level‐3 containment.  Reusable 
clothing should be laundered on‐site or by a laundering service. Personnel should not 
launder laboratory clothing at home.  
 
Gloves: Gloves should always be worn when handling biohazardous materials. 
Disposable gloves can provide an adequate barrier between the lab employee and most 
biohazardous materials.  
 
Respirators: When engineering controls (i.e. BSCs) are not available to provide adequate 
protection against aerosolized agents or when mandated by federal regulations, 
respirators shall be worn. Duke’s Respiratory Protection Program requires that 
employees be medically cleared, fit‐tested, and trained on proper usage and care before 
allowed to wear a respirator. Details of the Program can be viewed here. 
 
Disposable Shoe‐covers/Booties: When significant splash and splatter are anticipated, 
shoe‐covers/booties should be considered.  Prior to exiting the laboratory, these must 
be removed and disposed of properly. 
 
 Section 1 Biological Safety 
Chapter 3 
Standard Laboratory Practice and Technique 
Handwashing  
Hands should be washed as soon as possible when they come in contact with potentially 
infectious materials. A vigorous handwashing with a mild soap for 20 full seconds is 
appropriate. Hands should also be washed as soon as feasible after gloves are removed, and 
before exiting the laboratory.  
 
Eating, Drinking, Smoking, Applying Cosmetics and Handling Contact Lenses  
Eating, drinking, smoking, applying cosmetics and handling contact lenses is prohibited in work 
areas in which potentially infectious materials are being manipulated. Food and drink must not 
be stored in refrigerators in which laboratory materials are kept.  
 
Housekeeping  
Good housekeeping in laboratories can reduce the risk of accidents occurring. Work benches 
should be kept as clutter‐free as feasible, and aisles should always be free of trip hazards. 
Benches should be wiped down with an approved disinfectant at least once a day and 
immediately after a spill of potentially infectious materials.  
 
Pipetting  
Pipetting infectious agents can lead to personnel exposures by inhalation, contact, or ingestion 
if not performed properly. The following are a few safety precautions to be followed when 
pipetting in the laboratory:  1) Never mouth pipette; pipetting aids should always be used, 2) 
Pipette contents should be allowed to run down the wall of the container, making sure not to 
release the contents from a height, 3) Place absorbent paper on benchtops to reduce the risk of 
aerosols being generated by accidental dripping of infectious materials from pipette tips, and 4) 
Place disposable pipettes into pipette disposal boxes which have been lined with an autoclave 
bag, and then steam sterilize (autoclave) for 90 minutes at 121 degrees Celsius (see Waste 
Management Section).  
 
Sharps  
The use of needles, glass pipettes, glass slides and cover slips, scalpels and lancets should 
be eliminated, when possible. Appropriate precautions should be taken to avoid percutaneous 
injuries. These items should be disposed of immediately after use by placing them in an 
appropriate puncture‐resistant container. Bending, recapping or clipping of needles is 
prohibited. If recapping is absolutely necessary, a mechanical device or the one handed scoop 
method must be used. Plasticware should be used whenever possible, such as plastic graduated 
cylinders, funnels, aspirators, etc. Safety devices (i.e. mylar‐coated capillary tubes, Eclipse 
safety needles) should be used when available.  
 
 Section 1 Biological Safety 
Chapter 3 
Standard Laboratory Practice and Technique 
Decontamination  
The purpose of decontamination is to make a hazardous material safe for further handling. A 
decontamination procedure can range from sterilization to simple cleaning with soap and 
water. The following includes a description of the four main categories of physical and chemical 
means of decontamination. 
Heat: Wet heat is the most dependable method of sterilization. Steam autoclaving is the 
most convenient method available to the Duke laboratories for decontaminating 
biological waste and sterilizing glassware and media.  Note: Autoclaves that are used for 
decontamination of biohazardous wastes should be monitored for the efficacy of 
treatment.  This is accomplished by the use of biological indicators (i.e. spore strips).  
The generator of the waste (the lab) is responsible for performing and documenting this 
testing.  
Liquid Disinfection: Many types of liquid disinfectants are available under a variety of 
trade names. The most practical use of liquid disinfectants is for surface 
decontamination. Agents included in the category include, but are not limited to, 
quaternary ammonium compounds, phenolic compounds, halogens, aldehydes, alcohols 
and amines. A tuberculocidal disinfectant or diluted household bleach should always 
be used for decontamination when human materials are handled.  
 
NOTE: When household bleach is used for the decontamination of spills, a fresh solution (at 
least 10% household bleach) must be prepared.  Bleach solutions used for routine surface 
decontamination must be made up at least weekly.  Each solution container must be labeled 
with either a made‐on or an expiration date. 
 
Vapors and Gases: The use of vapors and gases as decontamination methods usually 
involve the decontamination of biological safety cabinets, but can also be used for 
whole building or room decontaminations. Agents used in this category include ethylene 
oxide, formaldehyde, gas, hydrogen peroxide and peracetic acid.  
Radiation: Ultraviolet radiation (UV) is sometimes used in biological safety cabinets for 
inactivating contaminants, but because of the low penetrating power of UV, dusty or 
soiled areas may limit its usefulness in the laboratory. Because UV can cause serious 
burns to eyes and skin, it must not be used when work areas are occupied. Whole room 
UV is not recommended. Do not rely on just radiation for your disinfection process.  
 
 Section 1 Biological Safety 
Chapter 3 
Standard Laboratory Practice and Technique 
 
Decontaminants and Their Use in Laboratories  
Decontaminant   Active  Temp  Contact  Vegetative  Lipo  Tubercle  Hydrophilic  Bacterial 
Ingredient/   (°C)   time   bacteria   viruses  bacilli   viruses   spores  
Concentration  (min.)  
Autoclave  Steam  121  50–90  +  +  +  +   +  
649‐
Incinerator  Heat  1‐60   +   +   +   +   +  
929  
Phenolic   0.2‐3%      10‐30  +  +  +  +   _  
 compounds 
Chlorine   0.01‐5%     10‐30  +  +  +  +   +  
 compounds 
Alcohol (ethyl or  70‐85%      10‐30  +  +  +  +   +  
isopropyl) 
*Formaldehyde  4‐8%      10‐30  +  +  +  +   +  
*Gluteraldeyhyde  2%     10‐600  +  +  +  +   +  
Hydrogen  
6%     10‐600  +   +   +   +   +  
 peroxide 
+ very positive response 
+ less positive response 
— negative response 
*irritating  characteristics of  agent precludes use for routine spill cleanup 
 
 Section 1 Biological Safety
Chapter 3
Standard Laboratory Practice and Technique

STANDARD LABORATORY PRACTICE AND TECHNIQUE

Biohazard Warning Signage

A sign incorporating the universal biohazard symbol must be posted at the entrance to the
laboratory when infectious agents are present.

Biosafety Level 1: The sign may include the name of the agent (s) in use, and the name
and phone number of the laboratory supervisor or other responsible personnel.

Biosafety Level 2: Posted information must include the name of the agent (s),
laboratory’s biosafety level, supervisor’s name (or other responsible personnel),
telephone number, and required procedures for entering and exiting the laboratory.

Biosafety Level 3: Posted information must include the name of the agent (s),
laboratory’s biosafety level, supervisor’s name (or other responsible personnel),
telephone number(s), and required procedures for entering and exiting the laboratory.

Personal Protective Equipment

Once a biological hazard has been identified, the supervisor and employee must agree on
the appropriate personal protective equipment (PPE) to be worn as the primary barrier of
protection. PPE may include, but is not limited to face protection, lab coats and gowns,
respirators, and shoe-covers/booties. Supervisory personnel are responsible for the initial
demonstration and periodic follow-up of proper use. Appropriate PPE should be donned before
handling potentially hazardous biological materials and removed immediately and replaced if
gross contamination of the equipment occurs. PPE should be removed before exiting the
laboratory.

Face Protection: When splash or splatter of infectious substances or other biological

materials is anticipated, appropriate face protection should be worn if work is
performed outside a biological safety cabinet. Such equipment would include but is not
limited to goggles, side-shielded safety glasses and chin length face shields.

Lab Coats and Gowns: Long sleeved lab coats or gowns should be worn to protect skin
and street clothes from contamination. In circumstances when splash or splatter is
anticipated, the garment must be resistant to liquid penetration. A cuffed lab coat or
gown should be worn when working with potentially infectious materials, and MUST be
worn when working with agents requiring Biosafety Level-3 containment. Reusable
clothing should be laundered on-site or by a laundering service. Personnel should not
launder laboratory clothing at home.
 Section 1 Biological Safety
Chapter 3
Standard Laboratory Practice and Technique

Gloves: Gloves should always be worn when handling biohazardous materials.

Disposable gloves can provide an adequate barrier between the lab employee and most
biohazardous materials. Double gloves and/or cut-resistant gloves should be considered
when handling sharp items and biohazardous materials.

Respirators: When engineering controls (i.e. BSCs) are not available to provide adequate
protection against aerosolized agents or when mandated by federal regulations,
respirators shall be worn. Duke’s Respiratory Protection Program requires that
employees be medically cleared, fit-tested, and trained on proper usage and care before
allowed to wear a respirator. Details of the Program can be viewed here.

Disposable Shoe-covers/Booties: When significant splash and splatter are anticipated,

shoe-covers/booties should be considered. Prior to exiting the laboratory, these must
be removed and disposed of properly.

Handwashing
Hands should be washed as soon as possible when they come in contact with potentially
infectious materials. A vigorous handwashing with a mild soap for 20 full seconds is
appropriate. Hands should also be washed as soon as feasible after gloves are removed, and
before exiting the laboratory.

Eating, Drinking, Smoking, Applying Cosmetics and Handling Contact Lenses

Eating, drinking, smoking, applying cosmetics and handling contact lenses is prohibited in work
areas in which potentially infectious materials are being manipulated. Food and drink must not
be stored in refrigerators in which laboratory materials are kept.

Housekeeping
Good housekeeping in laboratories can reduce the risk of accidents occurring. Work benches
should be kept as clutter-free as feasible, and aisles should always be free of trip hazards.
Benches should be wiped down with an approved disinfectant at least once a day and
immediately after a spill of potentially infectious materials.

Pipetting
Pipetting infectious agents can lead to personnel exposures by inhalation, contact, or ingestion
if not performed properly. The following are a few safety precautions to be followed when
pipetting in the laboratory: 1) Never mouth pipette; pipetting aids should always be used, 2)
Pipette contents should be allowed to run down the wall of the container, making sure not to
release the contents from a height, 3) Place absorbent paper on benchtops to reduce the risk of
aerosols being generated by accidental dripping of infectious materials from pipette tips, and 4)
Place disposable pipettes into pipette disposal boxes which have been lined with an autoclave
bag, and then steam sterilize (autoclave) for 90 minutes at 121 degrees Celsius (see Waste
Management Section).
 Section 1 Biological Safety
Chapter 3
Standard Laboratory Practice and Technique

Sharps
The use of needles, glass pipettes, glass slides and cover slips, scalpels and lancets should
be eliminated, when possible. Appropriate precautions should be taken to avoid percutaneous
injuries. These items should be disposed of immediately after use by placing them in an
appropriate puncture-resistant container. Bending, recapping or clipping of needles is
prohibited. If recapping is absolutely necessary, a mechanical device or the one handed scoop
method must be used. Plasticware should be used whenever possible, such as plastic graduated
cylinders, funnels, aspirators, etc. Safety devices (i.e. mylar-coated capillary tubes, Eclipse
safety needles) should be used when available.

Decontamination
The purpose of decontamination is to make a hazardous material safe for further handling. A
decontamination procedure can range from sterilization to simple cleaning with soap and
water. The following includes a description of the four main categories of physical and chemical
means of decontamination.
Heat: Wet heat is the most dependable method of sterilization. Steam autoclaving is the
most convenient method available to the Duke laboratories for decontaminating
biological waste and sterilizing glassware and media. Note: Autoclaves that are used for
decontamination of biohazardous wastes should be monitored for the efficacy of
treatment. This is accomplished by the use of biological indicators (i.e. spore strips).
The generator of the waste (the lab) is responsible for performing and documenting this
testing.
Liquid Disinfection: Many types of liquid disinfectants are available under a variety of
trade names. The most practical use of liquid disinfectants is for surface
decontamination. Agents included in the category include, but are not limited to,
quaternary ammonium compounds, phenolic compounds, halogens, aldehydes, alcohols
and amines. A tuberculocidal disinfectant or diluted household bleach should always
be used for decontamination when human materials are handled.

NOTE: When household bleach is used for the decontamination of spills, a fresh solution (at
least 10% household bleach) must be prepared. Bleach solutions used for routine surface
decontamination must be made up at least weekly. Each solution container must be labeled
with either a made-on or an expiration date.

Vapors and Gases: The use of vapors and gases as decontamination methods usually
involve the decontamination of biological safety cabinets, but can also be used for
whole building or room decontaminations. Agents used in this category include ethylene
oxide, formaldehyde, gas, hydrogen peroxide and peracetic acid.
Radiation: Ultraviolet radiation (UV) is sometimes used in biological safety cabinets for
inactivating contaminants, but because of the low penetrating power of UV, dusty or
soiled areas may limit its usefulness in the laboratory. Because UV can cause serious
 Section 1 Biological Safety
Chapter 3
Standard Laboratory Practice and Technique
burns to eyes and skin, it must not be used when work areas are occupied. Whole room
UV is not recommended. Do not rely on just radiation for your disinfection process.

Decontaminants and Their Use in Laboratories

Decontaminant Active Temp Contact Vegetative Lipo Tubercle Hydrophilic Bacterial
Ingredient/ (°C) time bacteria viruses bacilli viruses spores
Concentration (min.)
Autoclave Steam 121 50–90 + + + + +
649-
Incinerator Heat 1-60 + + + + +
929
Phenolic 0.2-3% 10-30 + + + + _
compounds
Chlorine 0.01-5% 10-30 + + + + +
compounds
Alcohol (ethyl or 70-85% 10-30 + + + + +
isopropyl)
*Formaldehyde 4-8% 10-30 + + + + +
*Gluteraldeyhyde 2% 10-600 + + + + +
Hydrogen
6% 10-600 + + + + +
peroxide
+ very positive response
+ less positive response
— negative response
*irritating characteristics of agent precludes use for routine spill cleanup
 Section 1 Biological Safety
Chapter 3
Standard Laboratory Practice and Technique

STANDARD LABORATORY PRACTICE AND TECHNIQUE

Personal Protective Equipment

Once a biological hazard has been identified, the supervisor and employee must agree on
the appropriate personal protective equipment (PPE) to be worn as the primary barrier of
protection. PPE may include, but is not limited to face protection, lab coats and gowns,
respirators, and shoe-covers/booties. Supervisory personnel are responsible for the initial
demonstration and periodic follow-up of proper use. Appropriate PPE should be donned before
handling potentially hazardous biological materials and removed immediately and replaced if
gross contamination of the equipment occurs. PPE should be removed before exiting the
laboratory.

Face Protection: When splash or splatter of infectious substances or other biological

materials is anticipated, appropriate face protection should be worn if work is
performed outside a biological safety cabinet. Such equipment would include but is not
limited to goggles, side-shielded safety glasses and chin length face shields.

Lab Coats and Gowns: Long sleeved lab coats or gowns should be worn to protect skin
and street clothes from contamination. In circumstances when splash or splatter is
anticipated, the garment must be resistant to liquid penetration. A cuffed lab coat or
gown should be worn when working with potentially infectious materials, and MUST be
worn when working with agents requiring Biosafety Level-3 containment. Reusable
clothing should be laundered on-site or by a laundering service. Personnel should not
launder laboratory clothing at home.

Gloves: Gloves should always be worn when handling biohazardous materials.

Disposable gloves can provide an adequate barrier between the lab employee and most
biohazardous materials. Double gloves and/or cut-resistant gloves should be considered
when handling sharp items and biohazardous materials.

Respirators: When engineering controls (i.e. BSCs) are not available to provide adequate
protection against aerosolized agents or when mandated by federal regulations,
respirators shall be worn. Duke’s Respiratory Protection Program requires that
employees be medically cleared, fit-tested, and trained on proper usage and care before
allowed to wear a respirator. Details of the Program can be viewed here.

Disposable Shoe-covers/Booties: When significant splash and splatter are anticipated,

shoe-covers/booties should be considered. Prior to exiting the laboratory, these must
be removed and disposed of properly.
 Section 1 Biological Safety 
Chapter 4 
Biohazard Spill Clean‐up 

BIOHAZARD SPILL CLEAN‐UP  
The following procedures should be followed to insure proper spill clean‐up:  
 
Spill Involving Blood or Body Fluids Wear disposable gloves. Absorb fluids with disposable 
towels. Clean area of all visible fluids with detergent (soap/water). Decontaminate area with a 
freshly prepared 1:10 dilution of bleach: water if surface is porous. If surface is hard and 
smooth use a 1:100 dilution.  Place all disposable materials into a plastic leak‐proof bag for 
disposal. 
   
Spill Involving Concentrated Microorganisms Requiring Biosafety Level 2 Containment  
(Staphylococcus sp., adenoviruses, etc.)  
Alert people in immediate area of spill. Put on appropriate protective equipment. Cover spill 
with paper towel or other absorbent materials. Carefully pour a freshly prepared 1:10 dilution 
of household bleach around the edges of the spill and then into the spill. Avoid splashing.  Allow 
a 20 minute contact period. Use paper towels to wipe up the spill, working from the outer 
edges into the center.  Clean spill area with fresh towels soaked in disinfectant. Discard 
disinfected disposal materials.  Alternatively, items that do not contain large amounts of bleach 
may autoclaved for 90 minutes at 121 degrees Celsius before disposal. 
 
Spill Involving Concentrated Microorganisms Required BSL 3 Containment  
(Mycobacterium tuberculosis, (TB) cultures)  
Attend to injured or contaminated persons and remove them from exposure. Alert people in 
the area to evacuate. Close doors to affected area; do not enter area for at least one hour. Have 
a person knowledgeable of the incident and area assist in proper clean‐up. Wearing gowns, 
gloves, respirator and shoe covers, clean up spills as indicated for Biosafety Level 2 organisms.  
   
 
 Section 1 Biological Safety 
Chapter 5 
Laundering of Laboratory Clothing 
 
LAUNDERING OF LABORATORY CLOTHING (i.e., lab coats)  
All laboratory clothing which is used as protective equipment should be laundered by the 
employer at no cost to employees. Soiled clothing being collected for laundering should be 
placed in leak‐proof container (e.g., biohazard bag). Soiled laundry should only be handled by 
individuals wearing appropriate PPE and should never be taken home. Reusable laboratory 
clothing worn in BSL‐3 areas must be decontaminated before being laundered.  
 
 Section 1 Biological Safety
Chapter 6
Waste Management

WASTE MANAGEMENT
Appropriate waste handling practices at Duke University and Medical Center are based on
compliance with OSHA regulations for protection of personnel who have to handle the waste,
and the North Carolina Medical Waste Regulations for appropriate disposal.

There are three primary methods for disposing of biological waste at Duke. These methods
include autoclaving, incineration, and chemical disinfection.

Autoclaving is usually the most convenient choice for labs since autoclaves are readily
available throughout most research laboratory buildings. Due to the closing of the City
of Durham Landfill, all landfill waste, including autoclaved laboratory waste, must
adhere to Virginia’s Medical Waste management Regulations.

Autoclaved Lab Waste Must Comply with VA State Law!

Red bags are NOT allowed in Virginia landfills. DO NOT use red bags, or they will be sent back to Duke.
Use ORANGE bags only. Orange autoclave bags are available for purchase in the VWR Stockroom or
through your preferred vendor. All biomedical waste must be autoclaved for at least 90 minutes at 250°F
(121°C) prior to disposal. Each bag must be tagged with a label which includes the date the bag was
autoclaved, the generator’s name, address and telephone number. Tags are available for purchase in the
VWR stockroom.

Incineration of biological waste is a viable option for all biological waste; however,
coordination with other departments is necessary to utilize this option. For pick-up of
biomedical lab waste, contact Environmental Services' Biomedical Waste Division for
pick-up (919-681-2727). The Division of Laboratory Animal Resources must be
contacted at 919-684-5212 for animal carcass disposal.

Chemical disinfection is a treatment option for liquid biological waste. An example is

household bleach.
 Section 1 Biological Safety
Chapter 7
Categories of Biological Waste and Acceptable Treatments

CATEGORIES OF BIOLOGICAL WASTE AND ACCEPTABLE TREATMENTS

Sharps — Needles, syringes with attached needles, capillary tubes, slides and cover slips,
scalpel blades, razor blades, and broken glassware that is contaminated with biological
material. These items should be placed in a puncture-resistant container (needlebox). There are
two acceptable methods for disposal of needleboxes; 1) place in orange autoclavable bags and
autoclaved before disposal or, 2) contact Environmental Services' Biomedical Waste Division for
pick-up (919-681-2727).

Pipettes — Pipette tips/serological pipette tips used to process human body fluids or cultures
of infectious agents, should be placed in a “pipette” box that is labeled with the biohazard
symbol and lined with an orange autoclavable bag. Once filled, these boxes should be placed in
orange autoclavable bags and autoclaved before disposal. Non-infectious pipettes should also
be placed in a puncture resistant container before disposal; however, it is not necessary to
autoclave.

Microbiological/Molecular Waste — Includes cultures and stocks of etiologic agents and

recombinant DNA/transgenics. Solid waste should be placed in orange autoclavable bags and
steam sterilized (autoclaved) before disposal. Liquid biological waste (no hazardous chemicals)
can be autoclaved or chemically treated (i.e. bleached) before disposal down the drain.

Specimens of human blood/body fluids that are OPIM — Containers of blood/body fluids less
than 20 mls and tissue cultures can be placed in orange autoclavable bags and autoclaved
before disposal.

Tissue Culture Wastes (Animal and Human) — All solid waste should be discarded in orange
autoclavable bags, and autoclaved before disposal. Liquid waste can be chemically disinfected
(bleach) before disposal down the drain.

Anatomical/Pathological Waste — Organs, limbs, animal carcasses etc., which must be

incinerated (Not Autoclaved!) for proper treatment. All large human-derived
anatomical/pathological waste should be submitted to Environmental Services' Biomedical
Waste Division. Animal carcasses should be disposed of through the Division of Laboratory
Animal Resources.

Non-contaminated glass —Items should be discarded in a lined heavy-duty cardboard box and
taped shut before disposal. Do NOT use cardboard boxes with “biohazard” symbols printed on
them, which implies biohazardous waste requiring special treatment.

Solid Disposal Supply Wastes — Disposable gloves, gauze, paper wrappings, parafilm, etc., that
are minimally contaminated. Decontamination is not required before disposal; however these
items should be placed in leakproof containers (i.e., a sturdy, plastic bag).
 Section 1 Biological Safety 
Chapter 8 
Biosafety Levels 
 
BIOSAFETY LEVELS Four biosafety levels (BSLs) are summarized in the table below for proper 
handling of biohazardous materials. BSLs consist of combinations of laboratory practices and 
techniques, safety equipment, and laboratory facilities. Each combination is specifically 
appropriate for the operations performed the documented or suspected routes of transmission 
of the infectious agents, and for the laboratory function tor activity.  
 
  
 
BSL  Agents  Practices  Safety Equipment   Facilities  
(Primary Barriers)  (Secondary Barriers) 
1  Not known to consistently  Standard  None required  Open bench top, sink required 
cause diseases in  microbiological 
immunocompetent adult  practices 
humans 
2  Associated with human  BSL‐1 practices plus:   Primary barriers: Class I or  BSL‐1plus:  
disease. Hazard:  • limited access   II biosafety cabinets or  • non‐fabric chairs and other 
percutaneous injury,  • biohazard warning  other physical  furniture easily cleanable  
mucous membrane  signs  containment devices used  • autoclave available  
exposure, ingestion  • sharps precautions   for all manipulations of  • eyewash readily available  
• biosafety manual  agents that cause splashes 
defining waste  or aerosols of infectious 
decontamination or  materials; PPE: laboratory 
medical surveillance  coats, gloves, face 
policies   protection as needed 
3  Indigenous or exotic agents  BSL‐2 practices plus:   Primary barriers: Class I or  BSL‐2 plus:  
with potential for aerosol  • controlled access   II biosafety cabinets or  • physical separation from access 
transmission; disease may  • decontamination of  other physical  corridors  
have serious or lethal  all wastes   containment devices used  • hands‐free handwashing‐ sink  
consequences  • decontamination of  for all manipulations of  • self‐closing double door access  
lab clothing before  agents; PPE: laboratory  • exhaust air not recirculated  
laundering   coats, gloves, respiratory  • negative airflow into laboratory  
• baseline serum   protection as needed  • eyewash readily available in lab  
4  Dangerous/exotic agents  BSL‐3 practices plus:   Primary barriers: All  BSL‐3 plus:  
which pose high risk of life‐ • clothing change  procedures conducted in  • separate building or isolated zone 
threatening disease,  before entering   Class III biosafety cabinets  • dedicated supply/exhaust, vacuum 
aerosol‐transmitted lab  • shower on exit   or Class I or II biosafety  and decon system  
infections; or related  • all material  cabinets in combination   
agents with unknown risk  decontaminated on  with full‐body, air supplied  There are no BSL‐4 labs at Duke 
of transmission  exit from facility   positive pressure suit 
Summarized from Biosafety in Microbiological and Biomedical Laboratories, 5th Edition, 2007. 
http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm 
 
Classification of Agents According to Risk  
Biological agents are assigned to biosafety levels (BSL) based on the risk they pose to human 
health and the environment. Such factors as severity of disease caused by the agent routes of 
exposure, and virulence are used when determining the most appropriate BSL. The partial list 
below is provided to assist laboratories in making preliminary decisions on the appropriate  
 Section 1 Biological Safety 
Chapter 8 
Biosafety Levels 
 
biosafety level for particular agents. Ultimately, the Occupational and Environmental Safety 
Office (OESO) will make the final BSL assignment. If a particular agent is not listed below, or if 
further assistance is needed in interpreting BSL requirements, contact the OESO‐Biological 
Safety Division at 684‐8822. There are no Biosafety Level 4 labs at Duke. 
 
Biohazard Warning Signage 
A sign incorporating the universal biohazard symbol must be posted at the entrance to the 
laboratory when infectious agents are present. 
 
Biosafety Level 1 (BSL‐1): The sign may include the name of the agent (s) in use, and the name 
and phone number of the laboratory supervisor or other responsible personnel.  
 
BSL‐1 is suitable for work involving well‐characterized agents not known to consistently 
cause disease in immunocompetent adult humans, and present minimal potential 
hazard to laboratory personnel and the environment. All bacterial, parasitic, fungal, 
viral, rickettsial, and chlamydial agents which have been assessed for risk but do not 
belong to a higher risk group can be safely handled at BSL‐1. Be aware that many agents 
not ordinarily associated with disease are opportunistic pathogens and may cause 
infection in the young, the aged and immunocompromised individuals. Examples of 
agents handled at BSL‐1 include: Bacillus subtilis, Eschericia coli ‐K12, Naegleria gruberi, 
etc.  
 
Biosafety Level 2 (BSL‐2): Posted information on the sign must include the name of the 
agent(s), laboratory’s biosafety level, supervisor’s name (or other responsible personnel), 
telephone number, and required procedures for entering and exiting the laboratory. 
 
BSL‐2 builds upon BSL‐1. BSL‐2 is suitable for work involving agents that pose moderate 
hazards to personnel and the environment. It differs from BSL‐1 in that: 1) laboratory 
personnel have specific training in handling pathogenic agents and are supervised by 
scientists competent in handling infectious agents and associated procedures; 2) access 
to the laboratory is restricted when work is being conducted; and 3) all procedures in 
which infectious aerosols or splashes may be created are conducted in BSCs or other 
physical containment equipment. 
 
BSL‐2 Viral Agents:  
Adenovirus   HTLV types I and II  
Creutzfeld‐Jacob agent   Human Blood & Blood Products  
Cytomegalovirus    Kuru  
Eastern equine encephalitis    Monkeypox virus  
Epstein‐Barr virus    SIV  
Hepatitis A, B, C, D, E    Spongiform encephalopathies  
Herpes simplex viruses    Vaccinia virus  
 Section 1 Biological Safety 
Chapter 8 
Biosafety Levels 
HIV   VSV (lab adapted strains) 
 
BSL‐2 Bacterial/Rickettsial Agents:  
Campylocacter fetus, coli, jejuni   Shigella boydii, dysenteriae,  
Chlamydia psittaci, trachomatis    flexneri, sonnei  
Clostridium botulinum, tetani   Treponema pallidum  
Corynebacterium diphtheriae     
Legionella spp    Vibrio cholera  
Neisseria gonorrhoeae     (including El Tor)  
Neisseria meningitidis    Vibrio parahemolyticus  
Pseudomonas pseudomallei    Vibrio vulnificus  
Salmonella spp  Yersinia pestis 
 
BSL‐2 Fungal Agents:  
Blastomyces dermatitidis   
Fonsecaea pedrosoi  
Cryptococcus neoformans   
Sporothrix schenkii  
Microsporum spp   
Trichophyton spp 
Exophiala dermatitidis (wangiella) 
 
BSL‐2 Parasitic Agents:  
Entomeoeba histolytia   
Strongyloides spp  
Crytosporidium spp   
Tania solium  
Giardia spp   
Toxoplasma spp  
Naegleria fowleri   
Trypanosoma spp 
Plasmodium spp 
 
Biosafety Level 3 (BSL‐3): Posted information on the sign must include the name of the 
agent(s), laboratory’s biosafety level, supervisor’s name (or other responsible personnel), 
telephone number(s), and required procedures for entering and exiting the laboratory.  
 
BSL‐3 is applicable to clinical, diagnostic, teaching, research, or production facilities where work 
is performed with indigenous or exotic agents that may cause serious or potentially lethal 
disease through the inhalation route of exposure. Laboratory personnel must receive specific 
training in handling pathogenic and potentially lethal agents, and must be supervised by 
scientists competent in handling infectious agents and associated procedures. All procedures 
involving the manipulation of infectious materials must be conducted within BSCs or other 
physical containment devices. A BSL‐3 laboratory has special engineering and design features. 
 
 
 Section 1 Biological Safety 
Chapter 8 
Biosafety Levels 
BSL‐3 Viral Agents:  
Valley    VSV exotic strains (Piry)  
Rift Valley Fever (Zinga)   Yellow fever (wild type) 
 
BSL‐3 Bacterial/Rickettsial Agents:  
Bacillus anthracis    Mycobacterium bovis  
Francisella tularensis    Rickettsia rickettsii  
Mycobacterium tuberculosis   Yersenia pestis (resistant strains) 
 
BSL‐3 Fungal Agents:    
Coccidioides immitis   Histoplasma capsulatum 
 
 
 Section 1 Biological Safety
Chapter 9
Laboratory Equipment
LABORATORY EQUIPMENT

Biological Safety Cabinets (BSCs)

BSCs are the most commonly used primary containment devices in microbiological laboratories.
There are three classes of BSCs (Class I, II, and III). When combined with appropriate
microbiological techniques, each Class provides different levels of protection.
Class I BSC — provide both personnel and environmental protection, however, they do
not provide product protection such as that needed for sterile tissue culture work. Class
I BSCs are suitable for work with low to moderate risk agents.
Class II BSC —these are the most commonly used BSCs at Duke. Class II BSCs provide
environmental, personnel and product protection. The main difference between Class I
and II cabinets is the HEPA filtration of the air flow down across the work surface of a
Class II cabinet.

Class II, Type A BSC

A. Blower
B. Rear plenum
C. Supply HEPA filter
D. Exhaust
E. Sash
F. Work surface

Things to Remember When Using a Class II BSC

Keep front and rear perforated grills free of clutter. Cluttered grills
can cause a disruption of air flow which can compromise personnel,
environmental and product protection. Avoid sudden movements in
and out of the cabinet. Also, avoid installing BSCs near windows or
frequently used doorways. Each of these can disrupt airflow. Gas
burners should not be used in the BSC. The heat disrupts air flow, the
flame can damage the HEPA filter and gas can build up inside the
work space due to recirculation of air. Volatile chemicals and volatile
radionuclides should not be used unless approved by the
Occupational and Environment Safety Office. Don’t store items on
top of the cabinet. The HEPA filter could be damaged and the
balance of air flow could be disrupted. Do not eat, drink, and chew
gum or smoke near the cabinet. Doing this could result in ingestion
of hazardous materials. Wipe down the cabinet interior with a
surface disinfectant before and after all manipulations.
 Section 1 Biological Safety
Chapter 9
Laboratory Equipment

Class III BSC — Gas tight BSCs provide the highest level of environmental, personnel and
product protection. A Class III BSC, (also referred to as a glove box), provides a complete
physical barrier between the product and personnel. These cabinets are used for high
risk biological agents when absolute containment is required.
A high efficiency particulate air (HEPA) filter is the main functional unit of a BSC. The
HEPA filter is a device which removes particulates and microorganisms from the air.
These filters remove 99.97% of all particulates 0.3 microns in diameter and have a
greater efficiency for particles < or > 0.3 microns. HEPA filters are made of boron silicate
fiber sheets which are pleated to increase surface area. In order to direct the airflow in
the filter, aluminum baffles separate each pleat.

Certification of Biological Safety Cabinets

BSCs are to be certified by one of the Occupational and Environmental Safety Office’s
“approved vendors”. These vendors are National Sanitation Foundation certified, and have
demonstrated expertise in maintaining BSCs. For a list of approved certifiers, contact the OESO
Biological Safety Division at 919-684-8822.
All cabinets in which human materials, infectious agents, or other potentially infectious
materials are being used must be certified annually. Cabinets in which non-infectious materials
are manipulated (i.e. sterile tissue culture) should be certified at least every two years, but
annually is encouraged. All newly purchased or recently moved cabinets must be certified
before they can be used for any type of work.
Any cabinet being used for work with infectious agents with the potential for aerosol
transmission (i.e. vaccinia virus) must be decontaminated with formaldehyde gas prior to
maintenance or relocation of the cabinet.

Clean Benches
Horizontal laminar-flow clean benches are designed to protect the product from contamination
and should never be confused with BSCs! The near-sterile work area makes these devices good
for many applications in which the product does not pose a risk to the worker. Clean benches
are considered inappropriate for work with potentially infectious agents.

Centrifuges
Centrifuges (including microhematrocrit centrifuges) are commonly used in the laboratory
environment. Centrifuges must be properly used and maintained to ensure safe operation.
The following are suggested practices:

• Refer to the owner's manual for routine maintenance requirements.

• Perform a visual inspection prior to each use (note unusual cracks, irregularities or
wear).
• Verify proper loading of specimens to maintain balance.
• After starting, listen for unusual noises or vibrations until programmed speed is reached.
 Section 1 Biological Safety
Chapter 9
Laboratory Equipment
• Perform routine decontamination of interior surfaces using an appropriate disinfectant.
Immediate decon is required when visible contamination is noted.
• Prevent the release of aerosols when centrifuging infectious materials that are spread
via the aerosol route by using "safety devices", (i.e. sealed buckets, safety trunnion
cups, and sealed heads). Safety cups must be opened in a BSC after centrifuging such
materials to avoid the release of aerosols into the room.
• Spills should be addressed immediately by following established biological spill
procedures. Special precautions should be taken when broken glass or other sharps
may be involved.

Homogenizers and Blenders

These items are commonly used in laboratories, and both are considered producers of aerosols.
Safety sealed homogenizers and blenders are commercially available and should be used when
working with those agents known or suspected of being transmitted through aerosols. The
purpose of these items is to contain any aerosols created during work procedures. These safety
devices may be used on the open benchtop; however, they must be opened in a BSC. All non-
sealed devices must be used exclusively in a BSC.
 Section 1 Biological Safety
Chapter 10
Human Blood, Blood Products, Tissues, and Body Fluids

HUMAN BLOOD, BLOOD PRODUCTS, TISSUES AND BODY FLUIDS

In 1991, the Occupational Safety and Health Administration (OSHA) promulgated a standard to
minimize the risk for occupational exposure to bloodborne pathogens (e.g., HIV, Hepatitis B).
The regulation, titled Occupational Exposure to Bloodborne Pathogens mandates several
provisions for those working with materials that are human-derived such as human blood,
blood products, other bodily fluids and any unfixed tissues. The full text of the Duke University
Bloodborne Pathogen Exposure Control Plan, including a copy of the Bloodborne Pathogen
Standard can be found in the Appendices of this section. The Plan must be readily available to
all employees working with those materials mentioned above. This includes all employees
working with primary human cell lines, or human cell lines that have not been well-
characterized and tested for human pathogens. The following are a few highlights of the Plan.

Universal Precautions
Universal precautions are defined as handling all human blood, body fluids, and tissues as if
they are infectious. This calls for the use of appropriate protective measures to reduce or
eliminate the risk of occupational exposure.

Hepatitis B Vaccination
All employees working with human blood, blood products, fresh tissues or bodily fluids shall be
offered the Hepatitis B vaccine at no cost to them. If an employee should decline the vaccine,
they must sign a waiver which is kept on file in the Employee Occupational Health and Wellness
(EOHW). For more information about the vaccine, contact the EOHW at 919-684-3136.

EOHW Blood and Body Fluid Exposure Hotline

All occupational exposures to potentially infectious materials are to be reported immediately by
calling 115 if on campus and 919-684-8115 if off campus. An EOHW representative will discuss
with the employee the appropriate follow up of the exposure. It is important that exposures are
reported as soon after the incident as possible because some post exposure treatments are
considered time sensitive.

Safety Training
All employees who work with materials (primary and well-characterized human cells, tissues,
blood) covered by OSHA’s Bloodborne Pathogen Standard are to receive initial safety training
and annually thereafter. Bloodborne Pathogens, General Laboratory Safety, and Biosafety Level
2 (BSL2) are available as online training modules. On-site general laboratory safety training can
be requested. In fact, credit for this may be given for those who participate in the annual lab
safety audit. Laboratory-specific training is the responsibility of the Primary Investigator.
Written standard operating procedures (SOP) for agents used at BSL2 are required and
supplement this general lab safety manual for your lab-specific training.
 Section 1 Biological Safety
Chapter 11
Recombinant DNA

RECOMBINANT DNA (rDNA)

Since the inception of rDNA technology, scientists have been concerned over the possibility that
artificially constructed rDNA could be biologically hazardous if not handled appropriately or
released into the environment. These concerns prompted the development of the NIH
Guidelines on rDNA research in May of 1976. The most recent revision was published in
September of 2009 and is available for review at
http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Guidelines_Apr_02.htm.
Researchers at Duke University who construct and/or handle materials containing recombinant
DNA molecules must comply with the requirements of the National Institutes of Health (NIH)
Guidelines for Research Involving Recombinant DNA Molecules. The following information and
procedures are developed to assist Duke University researchers with the documentation of this
compliance.

Generally, experiments requiring the use of recombinant biological agents should be handled
under the same BSL requirements as the wild type agent. For example, handling of adenoviral
vectors should be performed under BSL 2 conditions.

NIH Guidelines for Research Involving rDNA molecules are applicable to all rDNA research
conducted or sponsored by an institution that receives any support for rDNA research from the
NIH. rDNA research at Duke must be registered with the Duke Institutional Biosafety
Committee (IBC) whether or not the Principal Investigator received funding from NIH for the
project.

All rDNA research receiving NIH funding through Duke University but conducted outside of the
US must be registered with the Duke IBC and comply with any rules of the host country. The
NIH Guidelines provide guidance for containment and safety practices of various categories of
rDNA research.

What is rDNA?
The NIH rDNA Guidelines defines rDNA as 1) Molecules that are constructed outside living cells
by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell
and 2) Molecules that result from the replication of those described in (1) above.

At Duke, rDNA work may include:

1. Plasmids and viral vectors
2. Any synthetic DNA or RNA
3. Any RNA produced from rDNA, including messenger RNA (mRNA), small interfering RNA
(siRNA), micro RNA (miRNA), etc.
4. Genetically-modified organisms (animals, plants, bacteria, viruses, fungi, etc.). This
includes creation, cross-breeding, or manipulation of transgenic animals and plants.
5. Any such material obtained from another researcher or source
 Section 1 Biological Safety
Chapter 11
Recombinant DNA

The Institutional Biosafety Committee (IBC)

The NIH rDNA Guidelines requires that an IBC be established at any institution receiving NIH
funding for rDNA research to oversee all rDNA research at that institution, and insure that such
work is compliant with the Guidelines.
The mission of the Duke University IBC is to:
1. ensure that all recombinant DNA research conducted at the institution or sponsored by
the institution is conducted in compliance with the National Institutes of Health
Recombinant DNA Guidelines, and
2. ensure that protocols of research involving Select Agents (defined by the Centers for
Disease Control and Prevention), including but not limited to recombinant DNA, are
reviewed and found to comply with all national, state, and local requirements
3. ensure that all laboratory work conducted at Biosafety Level 3 containment and/or
Animal Biosafety Level 3 is reviewed and found to comply with all national, state, and
local requirements

The Duke University IBC has responsibility for such research throughout the Duke Health
System, Medical Center, and University. IBC members are appointed by the Chancellor of
Health Affairs and the Provost of Duke University. The Duke Occupational and Environmental
Safety Office staff will support the IBC in carrying out its mission. The IBC is authorized to
inspect research facilities, approve research practices and procedures, and to take actions, such
as enforcement of cessation of laboratory or clinical research activities, in the event of an
unsafe workplace situation.

Principal Investigator (P.I.) Responsibilities

The P.I. makes an initial determination of the required levels of physical and biological
containment, and practices and procedures in accordance with the NIH Guidelines. Determine
whether or not your research must be registered with the IBC by completing the rDNA survey.
If your research is not exempt submit the appropriate paperwork for the proposed work (see
rDNA Registration Process below).

The Principle Investigator:

1. Is responsible for adherence to all requirements of the NIH Guidelines,
including required safety practices.
2. Submits an annual update of the continuing protocols to the IBC.
3. Trains all laboratory workers regarding the potential hazards of the work and
precautions to be taken.
4. Investigates and reports any significant problems or illnesses pertaining to
the operation and implementation of containment, or any adverse reactions
occurring during clinical studies to the Biological Safety Office for review by
the IBC.
 Section 1 Biological Safety
Chapter 11
Recombinant DNA

5. Ensures that all lab workers experiencing occupational exposures to rDNA

material will report such exposures to Employee Occupational Health and
Wellness (EOHW) and the IBC.
6. Complies with any shipping requirements for rDNA molecules.
7. Ensures that laboratory workers who work with animals involved in the work
participate in the Duke Health Surveillance for Animal Handlers.

The rDNA Registration Process - All research that is not exempt from compliance with the NIH
Guidelines for Research Involving rDNA Molecules must be registered with the IBC. Non-exempt
manipulation of recombinant DNA molecules includes, but is not limited to cross-breeding to
create a new strain of animal or plant, rDNA in viral vectors or human cells, and rDNA in clinical
human trials.
1. Review the table “Required Documents for Protocol Submission to the
Institutional Biosafety Committee”.
2. Submit the appropriate documents to the IBC. Templates are below.
a. rDNA form
b. SOP for BSL2/ABSL2 labs, and
c. Plasmid/Vector Table.
3. Gene Transfer/Human Subject: every human trial requires its own review by
the NIH OBA, the Duke Institutional Review Board and the IBC, even if the
same rDNA material is used in multiple trials. See guidance information here.

General Laboratory Procedures - Review the general laboratory procedures for biosafety and
rDNA work. These procedures include physical containment, standard practices and training.
The procedures can be found in
1. The CDC Guidelines on Biosafety in Molecular and Microbiological Laboratories
(BMBL5). The BMBL has general descriptions (Section IV) of appropriate laboratory
biosafety levels.
2. Appendix G of NIH Guidelines. The appendix provides rDNA-specific descriptions.

Recombinant DNA Waste Management - rDNA and transgenic organisms must be treated the
same as medical or infectious waste before disposal. Organisms must be rendered inviable
before disposal. See the waste management policy for more information.

Incident Response and Reporting - The NIH requires institutions to report incidents involving
rDNA materials including loss, theft, or release. This includes both NIH exempt and non-exempt
rDNA materials.
1. Report any loss, theft, or release involving rDNA materials to the Occupational and
Environmental Safety Office at 919-684-8822.
2. Report any human exposure to rDNA to Employee Occupational Health and Wellness
(EOHW, 919-684-8115) and to the Occupational and Environmental Safety Office (OESO,
919-684-8822). Complete the Report of Occupational Injury or Illness form.
 Section 1 Biological Safety
Chapter 11
Recombinant DNA

Training - A variety of training is essential to ensure good lab practices. OESO training is
available at the on-line training website.
1. General Laboratory Safety is relevant to all lab workers.
2. Lab-specific orientation and training is provided by the P.I.
3. Biosafety Level 2 (BSL2) training is for those who handle infectious material or other
potentially infectious material (OPIM) that poses a splash, splatter, or percutaneous
exposure hazard.
4. Bloodborne Pathogens training is required for those who handle materials of human
origin (i.e. primary and well-established cell lines).
5. Biosafety Level 3 (BSL3) training for higher containment
6. Plant containment training (Phytotron website)
7. Animal handlers training (IACUC website)
 Section 1 Biological Safety
Chapter 12
Select Agents and Toxins

SELECT AGENTS AND TOXINS

(OESO Select Agent Website: http://www.safety.duke.edu/BioSafety/selectagents.htm)

All Select Agents and Toxins (see Appendix A) are ordered and obtained through the OESO -
Biological Safety Division 919-684-8822.

Background

On December 13, 2002, new regulations were published to implement the Public Health
Security and Bioterrorism Preparedness and Response Act of 2002, Public Law 107-188.
The regulations apply to possession, use, and transfer of certain biological agents and toxins,
and to recombinant DNA experiments involving those agents and toxins which pose a threat to
public health and safety. The regulated materials are referred to as "select agents" and lists of
these potential bioterrorist agents have been developed by 21 governmental agencies. The lists
are segregated by their potential targets: humans, humans and animals, animals only, and
plants only.

How was the Select Agents and Toxin list determined?

CDC prepared the Select Agents list for 42 CFR 73 after receiving extensive input from scientists
representing 21 Federal government entities. The Health and Human Services (HHS) Secretary
considered the following criteria for establishing the list:
o The effect on human health of exposure to the agent or toxin;

o The degree of contagiousness of the agent or potency of the toxin and the
methods by which the agent or toxin is transferred to humans;

o The availability and effectiveness of pharmacotherapies and immunizations to

treat and prevent any illness resulting from infection by the agent or toxin.
Regulations:
• 42 CFR Parts 72 and 73, 42 CFR Part 1003 RIN 0920–AA09 Possession, Use and Transfer
of Select Agents and Toxins; Final Rule.
http://oig.hhs.gov/authorities/docs/05/032905FRselectagents.pdf This regulation
covers select agents which target humans.

• 7 CFR Part 331 and 9 CFR Part 121 Agricultural Bioterrorism Protection Act of 2002;
Possession, Use and Transfer of Biological Agents and Toxins; Final Rule.
http://www.aphis.usda.gov/programs/ag_selectagent/FinalRule3-18-05.pdf This
regulation includes the "overlap" list in the HHS regulation, as well as a list of select
agents affecting only animals and plants.
 Section 1 Biological Safety
Chapter 12
Select Agents and Toxins

Transfer, Receipt, and Storage of Select Agents

Responsibilities and Procedures

“Responsible Official” (RO):

The Final Rule requires that a RO be designated at each institution where select agents are
shipped, received, and/or possessed. The Director of the Biological Safety Division of the
Occupational and Environmental Safety Office shall serve as Duke’s RO. An alternate may be
designated by the RO if deemed necessary. The primary responsibility of the RO is to oversee
the registration of the laboratory with the CDC and/or the APHIS, and assure that all
requirements of compliance are met. The RO must give final approval of the facility registration
application prior to it being submitted to the applicable federal department.

Researcher:
The principal investigator (PI) is held responsible for assuring that he registers all possession,
transfer, and receipt of select agents through the Duke University Biological Safety Office. He is
also responsible for assuring that his laboratory fully complies with all prescribed safety policies
and procedures. Consequently, the PI must work closely with the RO to assure compliance with
this standard.

Compliance with the regulations requires that the “Responsible Official” obtain a permit for the
procurement, storage, and work with select agents, and that the Principal Investigator agrees to
conduct all activities as described in the permit application. The documentation required is
described in the regulations, and includes security plans, background checks on those
authorized to access select agents, laboratory inspections and inventory recordkeeping.

The CDC / APHIS have developed a helpful website that provides guidance on the Select Agent
Rule, helpful FAQs, Required Forms and Resources:

http://www.selectagents.gov/

How Do Researchers Register Select Agents?

Researchers planning on handling any of the listed Select Agents or Toxins (see Appendix A)
must contact the Duke University Responsible Official (currently the Director of Biological
Safety) to begin the registration process with the CDC or APHIS. Call 919-684-8822 for
assistance.
 Section 1 Biological Safety
Chapter 12
Select Agents and Toxins

What toxins are regulated as select agents and what quantities are exempt?

Work with the toxins listed below is regulated by the CDC/APHIS unless the aggregate amount
under the control of a principal investigator does not, at any time, exceed the amount specified.
Although only amounts greater than the maximum permissible limit must be registered with
CDC/APHIS, any amount of these toxins must be ordered through the OESO - Biological Safety
Division Select Agent Ordering Website:
(http://www.safety.duke.edu/BioSafety/selectagents.htm).

Maximum Permissible Limit

HHS Toxins Amount

Abrin 100 mg

Botulinum neurotoxins 0.5 mg

Clostridium perfringens epsilon toxin 100 mg

Conotoxin 100 mg

Diacetoxyscirpenol (DAS) 1000 mg

Ricin 100 mg

Saxitoxin 100 mg

Shiga-like ribosome inactivating proteins 100 mg

Shigatoxin 100 mg

Staphylococcal enterotoxins 5 mg

T-2 toxin 1000 mg

Tetrodotoxin 100 mg
 Section 1 Biological Safety
Appendix A Chapter 12
Select Agents and Toxins

HHS SELECT AGENTS AND TOXINS USDA SELECT AGENTS AND TOXINS
Abrin African horse sickness virus
Botulinum neurotoxins African swine fever virus
Botulinum neurotoxin producing species of Clostridium Akabane virus
Cercopithecine herpesvirus 1 (Herpes B virus) Avian influenza virus (highly pathogenic)
Clostridium perfringens epsilon toxin Bluetongue virus (exotic)
Coccidioides posadasii/Coccidioides immitis Bovine spongiform encephalopathy agent
Conotoxins Camel pox virus
Coxiella burnetii Classical swine fever virus
Crimean-Congo haemorrhagic fever virus Ehrlichia ruminantium (Heartwater)
Diacetoxyscirpenol Foot-and-mouth disease virus
Eastern Equine Encephalitis virus Goat pox virus
Ebola virus Japanese encephalitis virus
Francisella tularensis Lumpy skin disease virus
Lassa fever virus Malignant catarrhal fever virus
Marburg virus (Alcelaphine herpesvirus type 1)
Monkeypox virus Menangle virus
Reconstructed replication competent forms of the 1918 Mycoplasma capricolum subspecies capripneumoniae
pandemic influenza virus containing any portion of the (contagious caprine pleuropneumonia)
Mycoplasma mycoides subspecies mycoides small
coding regions of all eight gene segments (Reconstructed
colony (MmmSC) (contagious bovine pleuropneumonia)
1918 Influenza virus)
Peste des petits ruminants virus
Ricin Rinderpest virus
Rickettsia prowazekii Sheep pox virus
Rickettsia rickettsii Swine vesicular disease virus
Saxitoxin Vesicular stomatitis virus (exotic): Indiana subtypes
Shiga-like ribosome inactivating proteins VSV-IN2, VSV-IN3
1

Shigatoxin Virulent Newcastle disease virus

South American Haemorrhagic Fever viruses
Flexal USDA PLANT PROTECTION AND QUARANTINE (PPQ)
Guanarito SELECT AGENTS AND TOXINS
Junin Peronosclerospora philippinensis (Peronosclerospora
Machupo sacchari)
Sabia Phoma glycinicola (formerly Pyrenochaeta glycines)
Staphylococcal enterotoxins Ralstonia solanacearum race 3, biovar 2
T-2 toxin Rathayibacter toxicus
Tetrodotoxin Sclerophthora rayssiae var zeae
Tick-borne encephalitis complex (flavi) viruses Synchytrium endobioticum
Central European Tick-borne encephalitis Xanthomonas oryzae
Far Eastern Tick-borne encephalitis Xylella fastidiosa (citrus variegated chlorosis strain)
Kyasanur Forest disease
Omsk Hemorrhagic Fever
Russian Spring and Summer encephalitis
Variola major virus (Smallpox virus)
I verify that I neither store nor use any of the
Variola minor virus (Alastrim)
select agents or toxins listed above.
Yersinia pestis

OVERLAP SELECT AGENTS AND TOXINS I verify that I store or use the following select
agents or toxins:
Bacillus anthracis
Brucella abortus ___________________________________
Brucella melitensis Agent/toxin; amount
Brucella suis
Burkholderia mallei (formerly Pseudomonas mallei)
Burkholderia pseudomallei (formerly Pseudomonas ___________________________________
pseudomallei) Name:
Hendra virus Lab:
Nipah virus Date:
Rift Valley fever virus
Venezuelan Equine Encephalitis virus

11/17/2008
1
A virulent Newcastle disease virus (avian paramyxovirus serotype 1) has an intracerebral pathogenicity index in day-old chicks (Gallus gallus) of
0.7 or greater or has an amino acid sequence at the fusion (F) protein cleavage site that is consistent with virulent strains of Newcastle disease
virus. A failure to detect a cleavage site that is consistent with virulent strains does not confirm the absence of a virulent virus.
 Section 1 Biological Safety
Chapter 13
Packaging and Shipping Biological Materials

PACKAGING AND SHIPPING BIOLOGICAL MATERIALS

Although several agencies have published regulations or guidelines for the proper packaging
and shipment of biological materials, the International Air Transport Association's (IATA)
Dangerous Goods Regulations (DGR) governs all international shipments. Furthermore, all air
transport of regulated biological materials (including domestic flights) must strictly adhere to
the DGR. For this reason, the OESO provided training is primarily focused on compliance with
these regulations.

Training
All personnel involved in the process of shipping biological materials must receive proper
training initially and at least every two years thereafter. Training is provided through the OESO
website's "online-training" link. The program is titled Shipping Biological Materials. The
Training Supplement Guide includes a summary of the most relevant training content for
properly classifying, packing and labeling a shipment. Note: The information provided in the
checklists below may not include all relevant shipping criteria, and are not intended to be used
without first completing the official training.
 Section 1 Biological Safety
Chapter 13
Packaging and Shipping Biological Materials

Checklist for Shipping Patient (animals and humans) Specimens (for which there is minimal
likelihood that pathogens are present)

Specimen Packaging
__ Specimen in leak-proof primary container
__ Absorbent material is sufficient to absorb entire contents of primary container(s)
__ Primary containers are wrapped individually
__ Leak- proof secondary container
Labeling Outer Container
__ Statement: “Exempt human specimen” or “Exempt animal specimen”
__ Miscellaneous Class 9 label(2) if shipment contains dry ice, "UN 1845" and amount used
in kg
Completing the Airbill
__ Name and address of shipper and recipient
__ Check “Saturday Delivery” box if applicable
__ In Section 6 (Special Handling) of the airbill, indicate that the shipment is NOT a
dangerous good
__ Check the “Dry Ice” box if applicable and indicate “UN 1845” and the quantity of dry ice
in kg
__ Shipper’s signature (optional)
Note: In determining whether a patient specimen has a minimal likelihood that pathogens are
present; an element of professional judgment is required. That judgment should be based on
the known medical history, symptoms and individual circumstances of the source, and endemic
local conditions.
 Section 1 Biological Safety
Chapter 13
Packaging and Shipping Biological Materials
Checklist for Shipping Biological Substance, Category B “Infectious Substances”

Specimen Packaging
__ Specimen in leak-proof primary container
__ Absorbent material is sufficient to absorb entire contents of primary container(s)
__ Primary containers are wrapped individually
__ Leak- proof secondary container
__ Itemized list of contents placed between secondary and outer container
Labeling Outer Container
__ UN 3373 label(1)
__ Statement: “Biological Substance, Category B” adjacent to UN 3373 label
__ Miscellaneous Class 9 label(2) if shipment contains dry ice, "UN 1845" and amount used
in kg
Completing the Airbill
__ Name and address of shipper and recipient
__ Check “Saturday Delivery” box if applicable
__ In Section 6 (Special Handling) of the airbill, indicate that the shipment is a dangerous
good, which does NOT require a Shipper’s Declaration
__ Check the “Dry Ice” box if applicable and indicate “UN 1845” and the quantity of dry ice
in kg
__ Shipper’s signature (optional)

(1) - (2) -
 Section 1 Biological Safety
Chapter 13
Packaging and Shipping Biological Materials

Checklist for Shipping Category A Infectious Substances

Specimen Packaging
__ Specimen in leak-proof primary container
__ Absorbent material is sufficient to absorb entire contents of primary container(s)
__ Primary containers are wrapped individually
__ Leakproof secondary container
__ Itemized list of contents placed between secondary and outer container

Labeling Outer Container

__ Infectious Substance, Class 6 label
__ “Infectious Substance Affecting Humans”, identification of agent in parentheses, “UN
2814” and net quantity of infectious substance
__ Miscellaneous Class 9 label if shipment contains dry ice, “UN 1845” and amount used in
kg
__ Name and telephone number of a responsible person
__ If shipment includes >50mL or 50g of a Category A infectious substance, then add a
“Danger, do not load in passenger aircraft” label to the outer container

Dangerous Goods Declaration Form

__ Name, address and phone number of shipper and recipient
__ Mark out non-applicable “Aircraft Box”
__ Mark out non-applicable “Radioactive” box
__ 24-hour emergency response telephone number
__ Name and title of signatory, place, and date
__ Shipper’s signature
“Nature and Quantity of Dangerous Goods” section of the Declaration Form
__ Complete this section using the information provided on Pages 5, 9 & 10 of the
Supplemental Training Guide
 Section 1 Biological Safety
Chapter 13
Packaging and Shipping Biological Materials

Importation of agents or vectors of human disease

Importation of infectious materials and vectors that may contain them is regulated by federal
law. When an infectious agent is being imported into the United States, it must be
accompanied by an importation permit which is issued by the United States Public Health
Service (USPHS). Permits are issued only to the importer who must be located in the United
States. Importation permit applications are available through the OESO-Biological Safety
Division. (919-684-8822), or the following: http://www.cdc.gov/od/ohs/biosfty/imprtper/htm
Shipping labels containing the universal biohazard symbol, the address of the importer, the
permit number and expiration date are issued to the importer with the permit. The importer
must send the labels and one or more copies of the permit to the shipper. The importation
permit, with the proper packaging and labeling, will expedite clearance of the package of
infectious materials through the USPHS Division of Quarantine and release by US Customs.

Importation of etiologic agents of animals, and plant pests

The United States Department of Agriculture's Animal and Plant Health Inspection Service
(APHIS) regulates the importation and domestic transfer of agents, which may pose a risk to
animals or plants. A permit must be obtained prior to the receipt of any material that could
pose a potential risk to animals or plants. The permitting procedures and forms are found at
the following: http://www.aphis.usda.gov/forms/index.html

Select Agents
The Department of Health and Human Services’ 42 CFR Part 73, titled Possession, Use, and
Transfer of Select Agents and Toxins, became law on February 7, 2002. All researchers who
possess or plan to possess select agents must be registered with the Centers for Disease
Control and Prevention. For a list of restricted agents and other Select Agent Program
requirements, see the following: http://www.cdc.gov/od/sap/
The Director of OESO's Biological Safety Division will serve as Duke's Responsible Official (RO)
for select agents. All CDC registrations must be facilitated through the RO. To contact the RO,
call 919-684-8822.
 Section 1 Biological Safety
Chapter 13
Packaging and Shipping Biological Materials

Proper Shipment of Non-Regulated Liquids

Provisions must be made to ensure that all non-regulated liquids (i.e. buffers, water, etc.) are
properly packaged to prevent leakage during transport. The packaging must be of good quality,
strong enough to withstand shocks normally encountered during transport. A triple-packaging
system similar to that prescribed for patient specimens (above) must be utilized. The following
must be met.

• Liquid is placed in a leak-proof primary container

• Absorbent material must be placed around the primary container (sufficient amount to
absorb entire contents of primary container
• Primary container(s) and absorbent material(s) are placed into leak-proof secondary
container.
• Inner packages (primary and secondary container) are placed into a sturdy outer
container (i.e. cardboard box). Cushioning material is added between secondary
container and outer shipper if deemed necessary.
 Section 1 Biological Safety
Chapter 14
References and Appendices

REFERENCES
Biological Safety: Principles and Practices; Fleming D, Hunt D, 4th ed., ASM, 2006
Biosafety in Microbiological and Biomedical Laboratories; 5th ed., CDC/NIH, 2007
Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety
Cabinets; 2nd ed., CDC/NIH, 2000
North Carolina Administrative Code 10G.1201-.1207, General Statute 130A-309.26, 1990
Virginia Regulated Medical Waste Department of Environmental Quality, Waste Management
Board Regulation: Title 9 VAC 20‐120, Regulated Medical Waste Management
Regulations; Effective June 19, 2002.
Occupational Exposure to Bloodborne Pathogens, Final Rule; 29CFR 1910.1030, OSHA, 1991
Occupational Health and Safety in the Care and Use of Research Animals; NRC, 1997
2010 Dangerous Good Regulations; International Air Transport Association, 51st Ed.
Possession, Use, and Transfer of Select Agents and Toxins; USDHHS, 42 CFR Part 73, 2009
North Carolina Administrative Code G.S. 130A-149, Biological Agent Registry, 2003

APPENDICES
OSHA Occupational Exposure to Bloodborne Pathogens Standard
Duke University Bloodborne Pathogens Exposure Control Plan
OESO Training Program
Duke University Institutional Biosafety Committee (IBC) Policies and Procedures
Recombinant DNA FAQs
Duke University's Select Agent Policy
Duke University Medical Waste Management Policy
National Select Agent Registry
 Section 2 Chemical Safety
Duke Chemical Hygiene Plan
Chapter 1
Introduction and Purpose

Laboratory Safety Manual

Section 2
Chemical Safety

Duke Chemical Hygiene Plan

INTRODUCTION
PURPOSE
All laboratories using hazardous chemicals are required to comply with the Occupational Safety and
Health Administration’s 29 CFR 1910.1450, Occupational Exposure to Hazardous Chemicals in
Laboratories. This standard requires that the employer develop a written Chemical Hygiene Plan (CHP),
which is capable of protecting employees from the health hazards associated with hazardous chemicals
in the laboratory.
This section of the Laboratory Safety Manual is Duke University's general CHP and is intended to
highlight general laboratory practices that are necessary for protecting workers from exposure to
hazardous chemicals. In addition, each laboratory will develop a written laboratory-specific chemical
hygiene plan that will be made available to all laboratory staff.

DEFINITIONS
Definitions for selected terms used in this policy are included below. Please see paragraph (b) of OSHA’s
Hazardous Chemicals in Laboratories Standard (29 CFR 1910.1450) for additional definitions related to
the chemical hygiene program.

Chemical Hygiene Plan – A written program developed and implemented by the employer which sets
forth procedures, equipment, personal protective equipment and work practices that (A) are
capable of protecting employees from the health hazards presented by hazardous chemicals
used in that particular workplace and (B) meets the requirements of paragraph (e) OSHA’s
Hazardous Chemicals in Laboratories Standard (29 CFR 1910.1450).

Chemical Hygiene Officer – An employee who is designated by the employer, and who is qualified by
training or experience, to provide technical guidance in the development and implementation of
the provisions of the Chemical Hygiene Plan.

Designated Area – An area which may be used for work with "select carcinogens," reproductive toxins
or substances which have a high degree of acute toxicity. A designated area may be the entire
laboratory, an area of a laboratory or a device such as a laboratory hood.
 Section 2 Chemical Safety
Duke Chemical Hygiene Plan
Chapter 1
Introduction and Purpose
Hazardous Chemical – Any chemical which is classified as a physical hazard or a health hazard, a simple
asphyxiant, combustible dust, pyrophoric gas, or hazard not otherwise classified.

Health hazard – Includes chemicals that are classified as posing one of the following hazardous effects:
acute toxicity (any route of exposure); skin corrosion or irritation; serious eye damage or eye
irritation; respiratory or skin sensitization; germ cell mutagenicity; carcinogenicity; reproductive
toxicity; specific organ toxicity (single or repeated exposure); or aspiration hazard. The criteria
for determining whether a chemical is classified as a health hazard are detailed in Appendix A of
the OSHA Hazard Communication Standard (HCS).
High Risk Procedures – These are procedures which are likely to require engineering controls beyond
those found in the standard laboratory; use of chemicals or toxins which require medical
surveillance, vaccination, special antidotes, or exposure monitoring; and operations that pose
significant risk of fire, explosion, or exposure to personnel if a malfunction were to occur (such
as a utility outage, runaway reaction, broken container, or chemical spill).

Laboratory - A facility where the "laboratory use of hazardous chemicals" occurs. It is a workplace where
relatively small quantities of hazardous chemicals are used on a non-production basis.

Medical Consultation – A consultation which takes place between an employee and a licensed health
care provider for the purpose of determining what medical examinations or procedures, if any,
are appropriate in cases where a significant exposure to a hazardous chemical may have taken
place.

Particularly Hazardous Substances – PHSs include common chemicals that are “select” carcinogens,
reproductive toxins, highly acute toxins, as well as substances that are highly reactive and/or are
included in EPA’s list of Extremely Hazardous Substances or the Department of Homeland
Security’s list of Chemicals of Interest.

Physical hazard – A chemical that is classified as posing one of the following hazardous effects:
explosive; flammable (gases, aerosols, liquids, or solids); oxidizer (liquid, solid, or gas); self-
reactive; pyrophoric (liquid, or solid); self-heating; organic peroxide; corrosive to metal; gas
under pressure; or in contact with water emits flammable gas; or combustible dust. The criteria
for determining whether a chemical is classified as a physical hazard are in Appendix B of the
Hazard Communication Standard.

Safety Data Sheets (SDSs) – Written or printed material concerning a hazardous chemical that is
prepared in accordance with the OSHA Hazard Communication Standard.
 Section 2 Chemical Safety
Duke Chemical Hygiene Plan
Chapter 2
Responsibilities

RESPONSIBILITIES

Department Heads shall:

• Ensure compliance with all requirements for chemical safety and hygiene within their respective
departments.
• Provide direction on the departmental approach to developing and implementing laboratory-
specific Chemical Hygiene Plans.
Principal Investigators (Research Labs)/Directors (Clinical Labs) shall designate a Laboratory Chemical
Hygiene Officer (CHO) and shall support the Laboratory CHO by:
• Ensuring compliance with all requirements for chemical safety and hygiene within the
laboratory or laboratories.
• Provide direction and support to the Laboratory CHO on implementing laboratory-specific
Chemical Hygiene Plans and Standard Operating Procedures.
• Review and approve laboratory-specific Standard Operating Procedures for all high-risk
procedures, including work with any reactive materials.
Laboratory Chemical Hygiene Officers shall develop and document the Laboratory-Specific Chemical
Hygiene Documentation for the lab, and ensure implementation (such as training and
coordinating audits). Responsibilities for the Laboratory Chemical Hygiene Officer are described
in the Laboratory-Specific Chemical Hygiene Documentation.
Laboratory Employees and Students shall plan and conduct laboratory operations in accordance with
this Chemical Hygiene Plan and the Laboratory-Specific Documentation. Responsibilities for
laboratory employees and students are described in the Laboratory-Specific Documentation.
The Occupational and Environmental Safety Office (OESO) shall:
• Review the Duke University Chemical Hygiene Program annually and update as needed.
• Develop and provide general laboratory safety training.
• Conduct exposure assessments and evaluate exposure control measures as necessary.
• Provide emergency response for chemical spills.
• Investigate laboratory accidents, including injuries and exposures.
• Maintain employee exposure records.
• Develop a list of chemicals which require additional control measures (Particularly Hazardous
Substances).
• Review and approve high-risk procedures.
• Conduct periodic safety audits of laboratories.
Employee Occupational Health and Wellness shall:
• Provide medical consultation and surveillance as needed.
• Provide medical care for employees who have been injured or exposed to hazardous agents in
the lab.
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PROCEDURES
LABORATORY-SPECIFIC CHEMICAL HYGIENE PLAN
Each laboratory shall develop written documentation of the following:
• The identity of the laboratory and names of the Department Head and Departmental Safety
Coordinator, the Principal Investigator or Director, and any other person responsible for
implementation of the site-specific chemical hygiene plan.
• The name of the chemical hygiene officer (or lab safety coordinator) for the lab.
• Contact information and emergency numbers for responsible parties and lab members.
• Emergency action plan.
• Inventory of Particularly Hazardous Substances. (A broader chemical inventory is encouraged.)
• Location of Safety Data Sheets (SDSs).
• Lab-specific Standard Operating Procedures (SOPs) for unique chemical hazards not covered by
this section and any highly hazardous chemicals and processes.
• The appendix for this section contains a list of SOPs and SOP templates available on our
Chemical SOPs and SOP Templates webpage. The SOPs on this website are considered part of
the University Chemical Hygiene Plan.
• Documentation of OESO approval for chemical High-Risk Procedures.
• Description of procedures for in-lab management of chemical waste disposal.
• Documentation of laboratory-specific training.
• Sign-off page to indicate that the CHP is accurate and has been reviewed (and updated as
needed) on an annual basis.

The above requirements can be met by completing our Laboratory-Specific Chemical Hygiene Plan
template.
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CHEMICAL HAZARD INFORMATION AND TRAINING

Required Hazard Awareness Training
General Lab Safety Training:
Each laboratory employee and student shall be required to take a general laboratory safety course
online before beginning work in the lab and annually thereafter.
Laboratory-Specific Training:
The Principal Investigator or Laboratory Safety Coordinator shall conduct laboratory-specific hazard
awareness training for each laboratory employee or student before that person begins working in the
lab. This training must cover all items specified in the laboratory-specific training checklist, included with
the Laboratory-Specific Chemical Hygiene Documentation. This hazard awareness training shall be
reviewed as necessary and any time a new hazard is introduced.

Required Sources of Chemical Hazard Information

Labels:
All containers of hazardous chemicals must be labeled with the following information: product
identifier; signal word; hazard statement(s); pictogram(s); precautionary statement(s); and name,
address, and telephone number of the chemical manufacturer, importer, or other responsible party.
Laboratories shall not remove or deface these labels. Chemical containers complying with the previous
version of the OSHA Hazard Communication Standard need not be re-labeled.
Containers that will be used only within the laboratory must be labeled, tagged, or marked with either:
• The required information from the label on the original container, or
• The product identifier AND words, pictures, symbols, or combination thereof, which provide at
least general information regarding the hazards of the chemicals and which, in conjunction with
the other information available, will provide employees with specific information regarding the
hazards of the chemical.
Safety Data Sheets (SDSs):
A Safety Data Sheet (SDS) is written or printed material concerning a hazardous chemical that is
prepared in accordance with the OSHA Hazard Communication Standard. SDSs must be obtained or
developed by all chemical manufacturers and importers for each hazardous chemical that they produce
or import. SDSs are available online for most major manufacturers; smaller manufacturers may
distribute paper copies with the product.
Labs must retain copies of any SDSs that they receive, and must provide employees with access to SDSs
for all chemicals in the laboratory. Labs may use the SDS resources available on the OESO website to
help them locate SDSs.
Electronic availability of these documents is an acceptable alternative to hardcopies only if the Principal
Investigator or Laboratory Safety Coordinator has ensured that all laboratory personnel have
demonstrated the ability to locate the necessary information and there is a backup means for obtaining
an SDS in the case of failure of the primary electronic system.

Inventory of Particularly Hazardous Substances:

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Each laboratory is required to maintain an inventory (list) of all Particularly Hazardous Substances.
Laboratories are encouraged to include other chemicals on the inventory as well.
Particularly Hazardous Substances are those chemicals which may present extreme risk potential to
laboratory workers if not handled appropriately; therefore, these substances may require additional
controls when used in the laboratory. A list of Particularly Hazardous Substances has been prepared by
the Occupational and Environmental Safety Office. This list includes common chemicals that are “select”
carcinogens, reproductive toxins, and highly acute toxins. This list also includes substances that are
highly reactive such as explosives, flammable solids, peroxide formers, oxidizers, and compounds that
are reactive with air or water.

Regulations, Polices, and Procedures:

Hazardous Chemicals in Laboratories Standard - The Occupational Safety and Health Administration
published this standard, which requires employers to inform laboratory employees of chemical hazards
and to limit employee exposures to these hazards. All laboratory employees must have access to this
standard.
Occupational Exposure Limits (OELs) – The OELs are airborne concentrations that have been determined
to be safe for employees for a set period of time. The Occupational Safety and Health Administration
(OSHA) has published Permissible Exposure Limits (PELs) for a number of chemicals, and the American
Conference of Governmental Industrial Hygienists (ACGIH), a professional organization, has published
Threshold Limit Values (TLVs). PELs and/or TLVs, or exposure limits published by other countries, may
be specified in the SDS. Employees must be familiar with exposure limits for the chemicals in use in the
lab.
Duke University Safety Manual – Laboratory employees must be familiar with the various policies in the
Duke University Safety Manual. Of particular relevance for chemical safety, the Hazardous Materials
chapter contains valuable definitions and guidance related to safe storage of hazardous materials,
including flammables, compressed gas cylinders, cryogens, corrosives, and liquefied petroleum gases.

Chemical Information for Materials Produced in the Laboratory:

Intra-laboratory use of chemicals of known composition – when a chemical of known composition is
produced and determined to be hazardous, the principal investigator or laboratory safety coordinator
must ensure that personnel who use this chemical are provided with appropriate training and controls.
Intra-laboratory use of chemicals of unknown composition – when a chemical of unknown composition is
produced in the laboratory, it must be considered a “Particularly Hazardous Substance” and handled
accordingly. Each investigator or laboratory supervisor has the responsibility to identify and characterize
these unknown chemicals as soon as possible so that it may be determined whether or not they are
hazardous.
Chemicals produced for inter-laboratory use – If a lab produces a chemical for distribution outside of the
laboratory, then all requirements of OSHA’s Hazard Communication Standard (29CFR1910.1200) must
be met. The provisions of this standard are presented in Duke’s Hazard Communication Policy and
include hazard determination, development of labels, and preparation of Safety Data Sheets.
Labs may contact the OESO Occupational Hygiene and Safety Division (919-684-5996) for additional
information about the required content of labels and SDSs.
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Recommended Chemical Safety Resources

Prudent Practices in the Laboratory – Published by the National Research Council, this book is an
essential resource for chemical hygiene and safety. Particularly useful are the Laboratory Chemical
Safety Summaries included for many common laboratory chemicals in Appendix B.
Safety in Academic Chemistry Laboratories – Published by The American Chemical Society, volume 1 (for
students) and volume 2 (for faculty and administrators) provide a basic overview on preventing
chemical-related accidents in the lab.
OESO website – There are a number of useful resources on the OESO webpage. Many of the links
relevant to laboratory employees are organized on the Laboratory Environment page under “Hazardous
Chemicals”. Note also that the OESO website provides access to toxicology information through the
Registry of Toxic Effects of Chemical Substances. To access this, go to the OESO SDS Resources page,
click on “CCOHS SDS Resources”, and then click on the “RTECS” link on the left side of the page.
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SAFE USE OF CHEMICALS

Overview of general strategy (hierarchy of controls)
The general strategy for keeping employees safe during work with chemicals (or other workplace
hazards) is to use a hierarchy of controls that places emphasis on keeping hazards out of the workplace
when possible. When use of hazardous chemicals is necessary, the preferred controls are those which
remove the hazard from the workplace or place a barrier between the worker and the hazard
(engineering controls) followed by work practices and personal protective equipment (PPE), which
require more effort on the part of the individual employee.

Elimination/substitution of hazards
When planning research or clinical laboratory activities, consider the hazards of the chemicals that will
be used. If possible, select an alternative procedure that uses less hazardous chemicals, or that
substitutes a less hazardous form of the same chemical. Here are some examples:
• Phosphate assay: Some phosphate assay methods require heating perchloric acid, which can
create explosive crystals in fume hood ductwork. Instead, use a method that does not call for
perchloric acid, or purchase a phosphate assay kit.
• Acrylamide gels: Acrylamide is a Particularly Hazardous Substance (possible human carcinogen).
Avoid potential exposure to acrylamide powder by purchasing precast polyacrylamide gels.
• Xylene: Consider using PARAclear or another environmentally-safe clearing agent instead of
xylene to reduce exposure and disposal concerns.
• Other examples: See OESO’s Safer Alternatives webpage for other recommended substitutions.

Obtaining Permission for Chemical High-Risk Procedures

When planning research involving chemical High-Risk Procedures (see definitions), the Laboratory
Chemical Hygiene Officer must obtain written permission from OESO before beginning work. Permission
must be requested by contacting the OESO-Laboratory Safety Program at 919-684-8822.
Some examples of chemical High Risk Procedures include use of the following:
• Large quantities of liquid nitrogen or other cryogens (OESO will assess ventilation) – For LN2,
“large” is more than one LN2 freezer and one attached Dewar per room, or filling a cryocart or
cooler, or LN2 piped in from a tank located outside the building);
• Hot, concentrated perchloric acid;
• Pyrophoric gases or other Particularly Hazardous Substances that are reactive;
• Botulinum toxin, tetanus toxin, or other toxins for which vaccination is recommended for
laboratory employees;
• Hydrofluoric acid, MPTP, or other chemicals for which an antidote or specific first aid treatment
is required; and
• Use of formaldehyde, methylene chloride, or other chemicals for which OSHA substance-specific
standards may require exposure monitoring or training.

In addition, written OESO permission will be required for any scale-up of a previously approved high-risk
procedure.
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Controlling Exposures
Engineering Controls:
Engineering Controls are designed to move an air contaminant away from employees and/or to create a
barrier between a hazard and the employee. Some common types engineering controls are discussed
below.
Chemical Fume Hoods are the primary containment devices used to protect personnel and the
laboratory environment from hazardous chemicals that may become airborne through volatilization or
aerosolization.
• Use a chemical fume hood when working with
o Particularly Hazardous Substances that are volatile or that are in powder form,
o Other volatile compounds,
o Chemicals with a strong odor, or
o Other materials as indicated by the chemical- or lab-specific Standard Operating
Procedure.
• Follow these work rules when working in a chemical fume hood:
o Make sure your fume hood has been certified within the last year. If not, contact your
maintenance provider so that they can arrange for certification (often through a
contractor).
o Check the air flow monitor before each use. It should show that the hood is under
negative pressure. (An alarm should sound if flow is too low.) See instructions below if
the hood is not functioning correctly.
o Keep the fume hood clear of clutter – only those materials necessary to the procedure
at hand should be placed inside the hood’s work space. Additional objects in the work
space may affect the hood’s air flow pattern and compromise employee safety.
o Keep the sash in the lowest practical position (and close the sash when hood is not in
use).
o Perform work tasks 6-8 inches behind the hood opening.
• A special note for working with perchloric acid – Using perchloric acid in a standard fume hood
can lead to accumulation of explosive perchlorate salts in the ductwork. Before using heated
(>150° C) OR concentrated (>85%) perchloric acid in any chemical fume hood, contact OESO at
919-684-8822 for approval.
• If your fume hood is not functioning properly, stop working in the hood, then close the sash and
label the hood to indicate that it is not working. Contact your maintenance provider to repair
the hood. If hood contents could create a hazardous situation in the room (even with the sash
down), leave the room and contact OESO at 919-684-2794.
Biological Safety Cabinets provide filtered air inside the cabinet, and filter the air that leaves the cabinet.
Though some biological safety cabinets are exhausted, their exhaust ducts may be under positive
pressure. These cabinets are primarily intended to protect employees from biological hazards and
should not be used for chemical hazards unless there is no chemical fume hood available and the use
has been approved by OESO.
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Local Exhaust ventilation can be used where there is a localized source of chemical vapors that can be
captured. Examples include snorkel-type exhaust and downdraft sinks. Local exhaust should only be
installed with the involvement of the facilities group for your building (Facilities Management
Department for University buildings, or Engineering and Operations for Medical Center buildings) and
with the approval of OESO.
Isolation devices physically separate a contaminant-generating process from the work environment.
These will often involve a sealed plexiglass box, and may be combined with local exhaust.
Process modification involves changing the temperature or pressure at which an experiment is
conducted, or using an inert gas or other change in the experimental procedure to reduce the likelihood
of exposure or other incident.

Procurement Controls:
Procurement Controls involve controlling employee exposures by making chemical purchasing decisions
that enhance employee safety. For example, labs should
• Order only needed amounts – Order an amount that will be used in the foreseeable future; don’t
order larger quantities for the bulk discount. Having a larger amount on hand means that there
is a greater potential for a harmful exposure (or, in the case of flammables, a fire), and may lead
to additional disposal costs in the future.
• Order a less hazardous form of the same chemical – Use the logic below to help choose the least
hazardous physical form that will work for your application.
o Dilute solutions are generally safer than more concentrated solutions.
o Aqueous solutions are generally safer to handle than powders requiring reconstitution.
o Pellets, tablets, granules, or flakes are generally safer to handle than powders.
• Purchase the chemical in a safer container – Order chemicals in shatter-resistant containers or
other containers that enhance employee safety.
o Shatter-resistant containers – When ordering corrosives or highly flammable chemicals,
choose containers that are less likely to break, such as metal, plastic, or PVC-coated
glass. These options will reduce the risk of exposure if the container is dropped.
o Pre-weighed vials with rubber septum – When ordering hazardous powders, consider
purchasing in a pre-weighed vial with a rubber septum. This eliminates the need to
handle the powder, as the diluent can be injected directly into the container.
• Check the existing inventory before ordering – Maintain a chemical inventory so that lab
members can check the availability of a chemical in the lab before ordering more.

Work Practice Controls:

General Practices
Housekeeping – General housekeeping is often overlooked, but is very important in keeping laboratory
employees safe.
• Never obstruct access to exits and emergency equipment such as eyewashes, drench hoses, and
safety showers.
• Remove unnecessary items, broken/unused equipment, and empty boxes and bottles from the
work area.
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• Keep cords and cables out of the work area and away from walkways. Use cable management
devices to bundle cords and cables together under desks and lab benches.
• Keep items off the floor so that your housekeeping service can clean effectively, and to reduce
the risk of trips and falls.
• Occasionally wipe down benchtop surfaces to avoid contaminating the work or employees’
clothing.
Working alone – Working alone with hazardous chemicals should be avoided if possible. If working alone
will be necessary, seek the approval of the principal investigator or laboratory safety coordinator, and
make sure that someone in the lab knows your plans.
Unattended Experiments – Experiments involving hazardous chemicals should not be left unattended,
but if circumstances require that the experiment run when the lab is not occupied, seek the approval of
the principal investigator or laboratory safety coordinator in advance.
Safe use of laboratory equipment –
• Use equipment only for its designated purpose.
• Be familiar with the manufacturer’s instructions before using.
• Inspect equipment for damage; do not use damaged equipment.
Care with sharps – Handle and store laboratory glassware with care to avoid damage; do not use
damaged glassware. Choose safe cutting devices – use of straight razors should be avoided in favor of
safety scalpels or other alternatives.
Manipulating chemicals – Plan your work to minimize hand (glove) contact with chemicals. Handle
chemicals in closed containers whenever possible, use care when pouring, and use tools that minimize
glove contact with the chemical. Where contact with chemicals is unavoidable, select gloves that are
impermeable to that chemical. You can consult one of the glove penetration guides found on OESO’s
webpage for Personal Protective Equipment.
Food and drink – Food and drink shall not be consumed or stored in areas used for laboratory
operations. If food and drink are to be allowed in the laboratory at all, there must be designated clean
areas and all employees must observe good hygiene in these areas (no gloves, no contaminated objects
moved from other parts of the lab into these areas).
Personal Hygiene and standard work attire –
• Wash hands before donning and after removing gloves, and any time that hands may be
contaminated. In order to avoid contaminating “clean” surfaces, remove gloves before handling
objects such as doorknobs, keyboards, telephones, and other objects that will be handled by
coworkers without gloves.
• Wash hands before eating, drinking, smoking, or applying cosmetics.
• Confine long hair and loose clothing.
• Wear closed-toed shoes and minimize exposed skin.
• If working with flammable materials, avoid wearing flammable clothing (such as many
synthetics).
• Wear standard personal protective equipment when in the lab and remove it before leaving the
laboratory area.
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Storage/Transport/Disposal:
Chemical storage –
• Store chemicals in quantities that are as low as practical.
• Make sure that all chemical containers are labeled. Commercial container labels should be
retained; containers for use only in the lab must be labeled with product identifier and words,
pictures, symbols, or combination thereof, which provide at least general information regarding
the hazards of the chemicals and which, in conjunction with the other information immediately
available to employees under the hazard communication program, will provide employees with
the specific information regarding the physical and health hazards of the hazardous chemical.
• Store chemicals in compatible storage groups, and away from incompatibles – compatibility
information should be included on the chemical’s SDS.
o For additional guidance, check the list of Compatible Storage Groups in Prudent
Practices in the Laboratory (National Research Council).
o Store acids and bases separately.
o Store flammables and oxidizers separately.
o Store corrosives away from metal (unless the metal has a corrosion-proof coating). Do
not store corrosives under the sink.
o Store water reactives away from sinks and water-based solutions.
• Follow the storage guidelines outlined in the Duke University Safety Manual Chapter on
Hazardous Materials for flammable and combustible liquids, compressed gas cylinders,
cryogens, corrosives, and liquefied petroleum gases.
• Use unbreakable secondary containers (such as bins or bottle jackets) for high-hazard materials
(including all PHSs).
• Periodically examine stored chemical containers for container and label integrity. Faded or
damaged labels should be replaced. Leaking or damaged containers should be disposed of
through OESO Environmental Programs (919-684-2794).

Transporting chemicals –
• Take precautions to avoid dropping or spilling chemicals. For example, observe the following
practices:
o When possible, have chemical purchases delivered directly to the laboratory.
o Make sure that chemical containers are sealed during transport.
o Carry breakable containers in specially-designed bottle carriers or leak-resistant,
unbreakable secondary containers.
o When transporting chemicals on a cart, use a cart that is suitable for the load and one
that has high edges to contain leaks or spills. The cart should be capable of negotiating
uneven surfaces without tipping the chemical container or the cart.
o Transport chemicals by traveling least-trafficked routes. When possible, use freight
elevators.
o Gas cylinders must be strapped to a hand truck specifically designed for that purpose.
Cylinder cover caps must be in place.
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• If transporting large amounts of chemicals for a laboratory move, (or any amount of chemicals
on a public road), contact the OESO Environmental Programs Division of OESO at 919-684-2794
for consultation on safe packaging and compliance with federal, state, and local laws.
• Shipping of chemicals must be done in compliance with all applicable federal, state, and local
laws. Contact the OESO Environmental Programs Division of OESO at 919-684-2794 for
consultation on safe packaging and compliance with these laws.

Managing chemical wastes –

• All laboratories that produce chemical waste must adhere to the requirements of Duke
University’s Chemical Waste Policy and the Laboratory Chemical Waste Management Practices.
These policies cover procedures to request and timelines for chemical waste pickup.
• For more information on becoming a registered chemical waste generator, contact OESO
Environmental Programs at 919-684-2794 and see the OESO Environmental Programs’ Waste
Management Services webpage.
• Chemicals shall never be disposed of in the trash or via sanitary sewer without the prior written
approval of OESO Environmental Programs.
• Avoid storing chemical wastes on the floor to reduce risk of spills. If storage on the floor is
unavoidable, place the waste bottle in secondary containment.

Administrative Controls:
Work removal – In some circumstances, it may be necessary to remove an employee from the
workplace, or restrict employees from performing specific laboratory tasks that may adversely affect
their health. In most cases, these restrictions will be related to employee sensitivity to chemicals or
allergens in the workplace. In very rare circumstances, restrictions or work removal may be required
because an employee has been overexposed to an OSHA-regulated material in the laboratory. In all
cases, Employee Occupational Health and Wellness will be involved in determining the specific
restrictions and/or work removal plan.
Scheduling – In some cases, scheduling may be used to reduce the intensity of exposure that any given
employee has to a particular task in the laboratory. Scheduling a variety of tasks in a day will help to
reduce ergonomic risk factors from static postures or repetitive motions. OESO does not recommend
limiting employees’ daily chemical exposures by rotating employees or spreading high-hazard work over
a longer time than would normally be used – instead, engineering controls and work practices should be
used to control chemical exposures.
Limiting access to the laboratory –
• Laboratory work areas with hazardous chemicals should be secured when unattended.
• If the laboratory plans to have visitors, they must be accompanied by laboratory personnel, and
the Duke Policy on Minors/Non-employees in Work Areas must be followed.

Personal Protective Equipment (PPE):

Laboratories must comply with the Personal Protective Equipment Policy in the Duke University Safety
Manual, including the requirement to complete PPE Hazard Assessments. In cases where PPE
requirements are documented as part of general guidelines (as below), or specific written Standard
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Operating Procedures (SOPs), there is no need to duplicate this documentation on a PPE Hazard
Assessment Form. However, in cases where there are unique hazards not addressed by the general
guidelines or written SOPs used by the lab, use of the PPE Hazard Assessment Form is required.
Employees must be trained on use of PPE as outlined in the PPE Policy, whether appropriate PPE is
documented in this policy, a chemical-specific SOP, on the PPE Hazard Assessment Form, or elsewhere.
See below for standard laboratory PPE and guidelines describing when to use additional PPE. Additional
information on PPE is available on the OESO PPE webpage.
Gloves – Disposable nitrile gloves (4 to 8 mils thick) should be worn when manipulating chemicals in
containers, but they are not appropriate for heavy contact with many laboratory chemicals. Nitrile
gloves offer better chemical protection than latex gloves, and eliminate the risk of latex sensitivity.
(Employees with sensitivity to nitrile should check with Employee Occupational Health and Wellness
and/or OESO for other glove recommendations.) NOTE: Replace gloves that have been exposed to
chemicals or that appear worn. In some situations, disposable nitrile gloves may not offer adequate
protection, including
• Use of acids or organic chemicals, or heavy contact with any chemicals – Standard nitrile gloves
offer good protection for dry chemicals and water-based solutions, but only limited protection
against some acids and many organic chemicals. These gloves are not appropriate for heavy
contact or total immersion with any chemicals. Use glove selection guides or manufacturer’s
recommendations to select a glove appropriate for the work being performed. This is especially
important for tasks involving extended glove contact with chemicals.
• Work with sharps or puncture/scratch hazards – If working with chemicals around glassware or
sharps, it is important to protect your hands (and your chemical gloves) from cuts and scratches
because non-intact skin is more vulnerable to chemical exposures. In these cases, it is advisable
to wear a cut-resistant glove over your chemical-resistant glove.
• Work with hot liquids or cryogens – see the protective clothing section for guidance.
Lab Coat/skin protection – Lab coats should be worn (buttoned) whenever there is a possibility of
splashes, spills, or other clothing contamination to lab personnel. Additional protective clothing may be
indicated in some situations, including:
• Risk of splash with corrosive materials or chemicals that may absorb through the skin - In
addition to the standard lab coat, impermeable aprons, sleeves and shoe coverings (or an
impermeable coverall), should be worn.
• Risk of splash with cryogens – In addition to the standard lab coat, wear an insulated apron and
cryogen gloves.
• Risk of splash with hot liquids – In addition to the standard lab coat, wear a rubberized apron
and heat-resistant impermeable gloves. Standard autoclave gloves are not appropriate for
handling hot liquids.
Eye/face protection – Safety glasses should be worn in the laboratory when chemicals are being used.
Safety goggles (not the same as safety glasses – see this link for a photograph of safety goggles) should
be worn whenever there is a risk of chemical splash, when working with glassware under reduced or
elevated pressures, when handling potentially explosive compounds, and when handling glassware or
liquids at high temperatures. If the risk of splash is high, a face shield should also be worn.
Closed-toed shoes – Closed-toed shoes should always be worn in laboratories.
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Respirators – In situations where the laboratory fume hood or local exhaust does not adequately
prevent inhalation exposure, respirators may be necessary. Use of respirators requires medical
clearance, annual training, and (in most cases) an annual fit test. For more information, contact the
OESO Occupational Hygiene and Safety Division at 919-684-5996 for more information.
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PREPARING FOR AND RESPONDING TO EMERGENCIES

Types of incidents
Each lab should consider the types of incidents that could have an adverse effect on people, research
efforts, property, and/or the environment and engage in planning efforts aimed at mitigating the impact
of the emergency (for example, arranging for critical laboratory equipment to be maintained on
emergency power) and on the necessary response for each situation.
Response actions
For possible chemical-related incidents, the lab should consider the response actions that will be
needed, such as use of spill pads, additional personal protective equipment, and emergency equipment,
as well as reliance on Duke-wide resources.
Written plan
Where a response will be needed at the time of an emergency, a written plan should be prepared by the
lab, describing the actions that will need to be taken.

Preparedness
Each lab should make sure that it has the equipment and other resources available to implement its
emergency plans. The following resources will be necessary for all research and clinical labs; if additional
resources are needed, the Laboratory Safety Coordinator and PI should identify those resources in the
written emergency plan, and make sure that they are available.
Emergency Response & Incident Reporting Guide
Each lab should post an up-to-date copy of the Duke Emergency Response and Emergency Reporting
Guide near the exit and/or primary telephone for the lab. This guide lists emergency contacts and
procedures for various types of incidents.
Laboratory Contact List
The Laboratory-Specific Chemical Hygiene Documentation must include an emergency contact list for
laboratory employees, especially the Principal Investigator and Laboratory Safety Coordinator.
Eyewashes, drench hoses, and safety showers
OSHA (29 CFR 1910.151(c)) requires that, “where the eyes or body of any person may be exposed to
injurious corrosive materials, suitable facilities for quick drenching or flushing of the eyes and body shall
be provided within the work area for immediate use.”
• See the Duke Safety Manual Policy on Emergency Eyewash and Shower Equipment for more
information on requirements for placement and testing of these emergency drench devices.
• It is very important for laboratory employees to be familiar with the location of the nearest
eyewash/drench hose unit and/or safety shower. This must be covered in laboratory-specific
training.
• Use the Weekly Eyewash Maintenance Log Sheet or other means to document that the weekly
check required in the Emergency Eyewash and Shower Policy has been completed.
Spill response kits
Spill kits with appropriate instructions, adsorbents, and protective equipment must be made available in
the laboratory so that laboratory employees may safely clean up a minor chemical spill. (Mercury spills
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are never considered minor – they always require OESO response.) It is the responsibility of the
Laboratory Safety Coordinator to ensure that it is stocked with needed supplies, and that all employees
know where the kit is stored and are trained on how to use it.
• Laboratory employees generally will not have respiratory protection available to them and
should not expect to clean up spills that involve hazardous concentrations of chemicals in the
air.
• Laboratory employees should be familiar with the hazards (including volatility) of the chemicals
they work with and should have a sense of the likely need for spill clean-up assistance from the
OESO Spill Response Team or other group, and how to contact available outside assistance (See
Emergency Response section below).
Fire extinguishers
See the Fire Safety Section of the Laboratory Safety Manual for information on the types of fires and
appropriate fire extinguishers. Labs using potentially flammable metals should contact OESO Fire Safety
for information on how to obtain an appropriate Class D extinguisher.
Antidotes
Some laboratory chemicals have acute exposure effects that may be relieved or minimized by an
antidote. The laboratory should work with Employee Occupational Health and Wellness (684-3136) to
determine if there are any counter-indications. For example, those who work with hydrofluoric acid
(HF) must stock calcium gluconate gel to be used as first aid in case of an HF burn. (Medical attention
should still be sought immediately for HF burns.)

Emergency Response
Most of the following information is also found in the Duke Emergency Response and Incident Reporting
Guide, which should be posted near the main exit and/or mail telephone for each lab.
Minor chemical spills (those that the laboratory staff is capable of handling without assistance)
• Alert people in the immediate area of the spill.
• Avoid breathing vapors from spill.
• Turn off ignition and heat sources if spilled material is flammable.
• Put on appropriate personal protective equipment, such as safety goggles, suitable gloves, and
long-sleeved lab coat.
• Confine spill to small area.
• Use appropriate kit to neutralize and absorb acids and bases.
• Use appropriate kit or spill pads for other chemicals.
• Collect residue, place in appropriate container, and dispose as chemical waste (call 919-684-
2794 for waste collection).
• Clean spill area with water.
Chemical spill on body
• Flood exposed area with running water from faucet, drench hose or safety shower for at least
15 minutes.
• Remove all contaminated clothing and shoes.
 Section 2 Chemical Safety
Duke Chemical Hygiene Plan
Chapter 3
Procedures

• If needed, obtain medical care from the nearest Employee Occupational Health and Wellness
location (call 919-684-3136 (or, after hours, 919-684-8115) for more information), or through
one of the other providers listed here.
• Report incident to supervisor and complete a Report of Work-related Accident, Injury or Illness.
Hazardous material splashed in the eye
• Immediately rinse eyeball and inner surface of eyelid with water continuously for 15 minutes.
• Forcibly hold eye open to effectively wash behind eyelids.
• Obtain medical attention.
• Report incident to supervisor and complete a Report of Work-related Accident, Injury or Illness.
Major chemical spills
• Alert people in the area to evacuate.
• Turn off ignition and heat sources if spilled material is flammable.
• On campus: Call 911 from a campus phone or 919-684-2444 from any phone. Off campus: Call
911.
• Attend to injured or contaminated persons and remove them from exposure.
• Have a person knowledgeable of the area assist emergency personnel.
Mercury spills
For spills on campus, call OESO Environmental Programs Division at 919-684-2794; after hours, call Duke
Police at 919-684-2444. For spills off campus, follow local procedures or call 911.
Contact numbers for assistance and information
• OESO Environmental Programs – 919-684-2794
• OESO Occupational Hygiene and Safety – 919-684-5996
Personal Injury
• All work-related injuries and illnesses, regardless of the severity, must be reported to the
supervisor.
• If needed, obtain medical care from the nearest Employee Occupational Health and Wellness
location (call 919-684-3136 for more information (after hours, call 919-684-8115), or through
one of the other providers listed here.
• Complete a Report of Work-related Accident, Injury or Illness.
• See the Workers’ Compensation website for additional information, or call Workers’
Compensation at 919-684-6693).
Fire
See your lab’s site-specific fire plan (a template is available in the Fire Safety section of the Laboratory
Safety Manual) for emergency procedures related to a fire.
Other incidents affecting property or the environment
For EMERGENCIES that may impact building integrity and/or harm people:
o Evacuate the immediate area. If the entire building needs to be evacuated, follow the
procedures in your Site-Specific Fire Plan.
 Section 2 Chemical Safety
Duke Chemical Hygiene Plan
Chapter 3
Procedures

o On campus: Call 911 from a campus phone or 919-684-2444 from any phone. Off
campus: Call 911.
For other incidents/accidents that do not pose immediate danger to people or the environment, call
919-684-2794 to report the incident. If maintenance support is needed, contact your maintenance
provider.
 Section 2 Chemical Safety
Duke Chemical Hygiene Plan
Chapter 3
Procedures

MEDICAL CONSULATIONS
General
Exposure-related medical examinations
All laboratory personnel exposed to hazardous chemicals are given the opportunity to receive a medical
examination or consultation under the following circumstances:
• Whenever an employee believes they have developed signs or symptoms associated with
exposure to a hazardous chemical.
• Whenever an employee is involved in a spill, leak, explosion, or accidental release during which
hazardous over-exposures may have occurred.
• Whenever occupational exposure monitoring indicates exposures above regulated levels.
Medical Consultations
Any employee may obtain a free medical consultation regarding concerns about chemical or other
occupational exposures by contacting Employee Occupational Health and Wellness at 919-684-3136.
Students would contact Student Health at 919-681-WELL (-9355).
Reproductive Health Consultation
It is the intent of Duke University to provide a laboratory work environment which compromises neither
the reproductive health of laboratory workers, regardless of gender, nor the health of the fetus. See the
Reproductive Health Policy in the Duke University Safety Manual or the OESO Reproductive Health
website for more information.

Chemical specific
Work with biologically-derived toxins
There are vaccines that can reduce susceptibility to some of the biologically derived toxins, including
botulinum toxin and tetanus toxin. There are also post-exposure regimens for some of these toxins.
Employees working with toxins that have available vaccines or post-exposure treatments must contact
Employee Occupational Health and Wellness (EOHW) at 919-684-3136 to discuss (and obtain, if desired)
the vaccine and/or to arrange for EOHW to have the post-exposure treatment on hand.
Work with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
Employees working with MPTP will need to undergo medical surveillance at EOHW (919-684-3136), and
will likely be prescribed selegiline to use in case of exposure (to help counteract the Parkinsonism
symptoms associated with MPTP exposure).
Work with other chemicals for which unusual and/or rapid post-exposure treatment may be needed
Laboratory employees working with other chemicals which may require post-exposure treatment should
consult with EOHW before beginning work to determine medical surveillance requirements and to make
other arrangements.

Respirator medical clearance

Respirator Medical Clearance Questionnaire
Employees wearing respirators will be asked to complete this questionnaire (required by OSHA). It will
be reviewed by a nurse. If significant health concerns are noted, the nurse may contact the employee
 Section 2 Chemical Safety
Duke Chemical Hygiene Plan
Chapter 3
Procedures

with questions, or may schedule a medical consultation/examination at EOHW for the employee.
Employees who wear respirators must contact EOHW if they experience a change in their medical
condition that may affect their ability to wear their respirator. For more information, see the Duke
Respiratory Protection Policy.
 Section 2 Chemical Safety
Duke Chemical Hygiene Plan
Chapter 4
Exposure Monitoring

EXPOSURE MONITORING
OESO will conduct hazard assessments and, if needed, exposure monitoring, if there is reason to believe
that exposure levels for a chemical may exceed regulatory limits. Employees concerned about possible
exposures should contact the OESO Occupational Hygiene and Safety Division at 919-684-5996. The
decision to conduct exposure monitoring shall be based on
• The hazard potential of the chemical,
• The amount of the chemical used,
• The type and effectiveness of control measures that are in place, and
• The presence of over-exposure indicators such as odor, visual appearance, or symptoms of
exposure.
 Section 2 Chemical Safety
Duke Chemical Hygiene Plan
Chapter 5
Appendix

APPENDIX - SPECIFIC REQUIREMENTS FOR CERTAIN CHEMICAL GROUPS

Standard Operating Procedures (SOPs) for specific chemicals and chemical groups listed on our SOPs and
SOP Templates website are considered part of the Duke University Chemical Hygiene Plan. Check the
website for the most current list of SOPs and templates, as it is updated on a regular basis.
 Section 3 Fire Safety
Chapter 1
Introduction and Purpose

Laboratory Safety Manual

Section 3
Fire Safety

INTRODUCTION

PURPOSE

Both flammable and combustible materials are commonplace in most Duke Laboratories. For
this reason, all laboratory employees should be aware of the risks in their work spaces and
understand how to respond appropriately should a fire occur. Employees who take the time to
familiarize themselves with the location of safety devices (i.e., fire extinguishers, pull alarms,
safety showers, fire blankets, etc.) and proper route of egress before an accident occurs are
more likely to respond to an emergency situation in a calm and efficient manner.

Duke employees are encouraged to implement the RACE acronym in the case of a fire. RACE
is defined as the following:

Remove all individuals from the affected area

Activate the pull alarm and dial 911
Close all doors and windows
Extinguish the fire
 Section 3 Fire Safety
Chapter 2
Safe Handling of Flammable Chemicals

Safe Handling of Flammable Chemicals

Laboratory personnel should know the properties of individual chemicals with which they work.
The more familiar one becomes with a chemical, the more likely he/she is to handle the
chemical in a prudent manner. There are numerous safety resources available to all employees
who may have questions regarding a particular chemical. If developed appropriately, the
laboratory’s chemical hygiene plan should contain written standard operating procedures for
those chemicals that pose a fire risk in the laboratory. Safety data sheets (SDSs) are available
from the manufacturer or through OESO’s website. OESO may be consulted on safety practices
for particular chemicals. The following are several practices which should always be
implemented when handling flammable chemicals:

• Flammable chemicals should be stored in appropriate areas within the

laboratory and away from any potentially incompatible materials (see Section
2, Chapter 3 and the SOP for Flammable Materials). Storage of flammable
chemicals outside an approved flammable storage cabinet should be kept to a
minimum.

• Purchases of flammable chemicals should be kept to a minimum.

• All sources of ignition (e.g., Bunsen burners, hot plates, and electrical
equipment) should be eliminated from areas in which flammable or
combustible chemicals are used.

• Use the chemical fume hood to capture vapors when appreciable quantities of
flammable substances are being used.

• Only those refrigerators and freezers approved for flammable storage should
be used for the storage of flammable materials.

• Keep containers of flammable chemicals closed at all times when not in use.
 Section 3 Fire Safety
Chapter 3
Defining Flammable and Combustible Liquids

Defining Flammable and Combustible Liquids

Flammable Liquids
The National Fire Protection Association defines flammable liquids as any liquid having a flash
point below 100°F and having a vapor pressure exceeding 2068.6 mm Hg (40 psia) at 100°F.
Class IA — flash point below 73°F and Boiling Point (B.P). at or below 100°F
Class IB — flash point below 73°F and B.P. above 100°F
Class IC — flash point at or above 73°F, but below 100°F

Combustible Liquids
The National Fire Protection Association defines combustible liquids as any liquid having a flash
point at or above 100°F
Class II — flash point at or above 100°F, but below 140°F.
Class IIIA — flash point at or above 140°F, but below 200°F.
Class IIIB — flash point at or above 200°F.

National Fire Protection Association (NFPA) System for Classification of Hazards

The NFPA diamond is a symbol used to identify the hazards associated with a given chemical to
rescue workers. Frequently this symbol is found on the sides of buildings where chemicals are
stored and on chemical containers. Below are shown the various hazards symbolized by the
diamond and the numerical code which indicates the severity of the hazard. Class IA & IB
flammable liquids have an NFPA Fire Hazard rating of 4. Class IC flammable liquids are rated as
3. Combustible liquids are rated as 1, or 2.

HEALTH HAZARD FIRE HAZARD

4 — Deadly Flash Points:
3 — Extreme Danger 4 — Below 73° F
2 — Hazardous 3 — Below 100° F
1 — Slightly Hazardous 2 — Above 100 F°, Not Exceeding 200° F
1 — Above 200° F
0 — Will not burn

SPECIFIC HAZARD REACTIVITY

Oxidizer OX 4 — May detonate
Acid ACID 3 — Shock and heat may detonate
Alkali ALK 2 — Violent chemical change
Corrosive CORR 1 — Unstable if heated
Use NO WATER 0 — Stable
Radioactive
 Section 3 Fire Safety
Chapter 3
Defining Flammable and Combustible Liquids

NFPA Fire Hazard Ratings of Some Common Laboratory Chemicals

NFPA Rating Flash Point (°C) Boiling Point (°C) Ignition Temp. (°C)
Acetaldehyde 4 -37.8 21.1 175
Acetic Acid (glacial) 2 39 118 463
Acetone 3 -18 5607 465
Acetonitrile 3 6 82 524
Carbon disulfide 3 -30.0 46.1 90
Cyclohexane 3 -20.0 81.7 245
Diethylamine 3 -23 57 312
Diethyl ether 4 -45.0 35.0 160
Dimethyl sulfoxide 1 95 189 215
Ethyl alcohol 3 12.8 78.3 365
Heptane 3 -3.9 98.3 204
Hexane 3 -21.7 68.9 225
Hydrogen 4 --- -252 500
Isopropyl alcohol 3 11.7 82.8 3
Methyl alcohol 3 11.1 64.9 385
Methyl ethyl ketone 3 -6.1 80 515
Pentane 4 -40.0 36.1 260
Styrene 3 32.2 146.1 490
Tetrahydrofuran 3 -14 66 321
Toluene 3 4.4 110 480
p-Xylene 3 27.2 138.3 530

Storage of Flammable and Combustible Liquids Outside of Flammable Storage Cabinets

The maximum quantity of Class I Flammable Liquids outside an approved storage cabinet shall
not exceed 2 gallons per 100 square feet of laboratory space.

The combined maximum quantity of Class I, II and III Flammable Liquids and Combustibles
outside an approved storage cabinet shall not exceed 5 gallons per 100 square feet of
laboratory space.
 Section 3 Fire Safety
Chapter 3
Defining Flammable and Combustible Liquids

Chemical containers, not actively being used, should not be stored in the work area of chemical
fume hoods. Too much clutter can disrupt air-flow patterns and potentially compromise worker
protection.

Storage of Flammable Liquids in Refrigerators and Freezers

Per enforcement directive from the City of Durham Fire Marshal, all laboratory refrigerators
and freezers must be labeled to indicate whether or not they are suitable for storing flammable
liquids.

Refrigerators and freezers utilized throughout the University, Hospital, and Medical Center
generally fall within the following three categories:

1. Those designed to store flammable liquids with all electrical equipment that meets Class I,
Division I requirements.

2. Those that have been modified by a licensed electrician to meet the Class I, Division I
requirements.

3. Those “residential-types” that cannot be utilized to store flammable liquids, but are used to
store other chemicals or laboratory reagents.

Those refrigerators and freezers which fall into either category 1 or 2 will require a blue and
white label which states that the device is approved for flammable storage. Those which fall
into category 3 will require a red and white label be affixed which states that the appliance is
not approved for flammable storage.

To request aid in identifying the category in which an appliance falls, contact the OESO Fire
Safety Division at 919-684-5609.
 Section 3 Fire Safety  
Chapter 4 
Classes of Fires 

Classes of Fires  

Class A fires are those which involve ordinary combustible materials such as wood, paper or 
cloth. These fires should be extinguished by using a dry chemical extinguisher. Water is 
effective in extinguishing these type fires, however, water extinguishers are rarely found in the 
Medical Center.  

Class B fires are those which involve flammable liquids, gases, oil, paint and greases. Either dry 
chemical or carbon dioxide extinguishers should be used to extinguish these type fires. Note: 
flammable liquids may re‐ignite after being extinguished. DO NOT USE WATER!  

Class C fires are those which involve electricity. Either dry chemical or carbon dioxide 
extinguishers should be used to extinguish these type fires. DO NOT USE WATER!  

Class D fires are those which involve combustible metals such as magnesium or sodium. Water 
can react with sodium and other alkali metals explosively, therefore DO NOT USE WATER! Also 
understand that CO2 extinguishers are unlikely to be able to contain a Class D fire.  

 
 Section 3 Fire Safety
Chapter 5
Fire Extinguishers

Fire Extinguishers

There are three basic types of portable fire extinguishers found throughout the Medical Center
and University. These include dry chemical, carbon dioxide and halotron extinguishers. These
devices are to be used to extinguish small or beginning fires. Any employee wishing to operate
an extinguisher should contact the OESO Fire Safety Division at 919-684-5609.

CO 2 Fire Extinguishers

The carbon dioxide extinguisher is rated to extinguish Class B and C fires. The carbon dioxide is
in the extinguisher as a liquid under pressure, and is discharged as a gas. Extinguishing is
accomplished by removing the oxygen from the fire. Carbon dioxide is a “clean” agent which
will evaporate and leave no residue.

Dry Chemical Extinguishers

Dry chemical extinguishers are intended for use on Class A, B or C fires. Best results are
obtained by attacking the near edge of the fire and progressing forward, moving the nozzle
rapidly with a side-to-side sweeping motion. Discharge should be continued after flames are
extinguished (especially on Class A fires) to prevent possible re-ignition.

Halotron Extinguishers

Halotron is a clean fire extinguishing agent which is a safe and environmentally acceptable
replacement for halon 1211. Halotron, which is discharged as a liquid which rapidly evaporates,
will be used throughout many patient care areas.

Class D Fire Extinguishers

These types of fire extinguishers are rarely used in the Medical Center and University.
Laboratories using potentially flammable metals should contact the Fire Safety Division at 919-
684-5609 for information about getting a Class D extinguisher. Extinguishers for Class D fires
must match the type of metal that is burning. Usually a list of metals for which the extinguisher
is appropriate is shown on the extinguisher’s labeling. Prudent Practices in the Laboratory:
Handling and Disposal of Chemicals recommends the use of Met-L-X(,or Met-L-Kyl)
extinguishers.
 Section 3 Fire Safety
Chapter 5
Fire Extinguishers

All employees should be familiar with the location of extinguishers in his or her work area. In
order to operate an extinguisher appropriately, one should implement the PASS acronym which
stands for:

P—Pull the pin

A—Aim the nozzle at the base of a fire
S—Squeeze the handle
S—Sweep the base of the fire
 Section 3 Fire Safety
Chapter 6
Fire Drills, Site-specific Fire Plan, and References

Fire Drills

Fire drills are conducted in all University, Hospital and Medical Center buildings by OESO Fire
Safety on a routine basis. If your laboratory hasn’t participated in a complete evacuation drill in
the past year, contact Fire Safety at 919-684-5609 to schedule a drill in your area.

Site-Specific Fire Plan

Each laboratory should develop a site-specific contingency plan in the event of a fire. The
following template is to be completed as accurately as possible and returned to the Fire Safety
Division at Box 90427. Once reviewed and approved, the plan will be mailed back to the
laboratory. The completed plan should be placed in an area where it is easily accessible by all
laboratory personnel.

Site Specific Fire Safety Plan

(Give Exact Name of Lab or Clinic)

The ___________________________ adheres to the general Hospital/Medical Center OESO

Fire Safety policy as found in the Duke University & Medical Center Safety Manual. All
personnel are responsible for the knowledge and compliance with Policy as well as their
departmental specific Policy described below.

1. Specific roles and responsibilities for personnel who are in the area of and away from a
fire’s point of origin;

• The laboratory is located in the ______________________ Building, room(s)

______________.
•

• There are __________________________________ exits from the laboratory which are

located
________________________________________________________________________
_______
•

• The evacuation plan for the laboratory is located

____________________________________ .
 Section 3 Fire Safety
Chapter 6
Fire Drills, Site-specific Fire Plan, and References

• The department’s specific zone is ____________________ .

The fire point is the area where the alarm is activated. Medical Center buildings use standard
fire alarm systems that produce an audible tone (horn, bell etc.). All staff members are
expected to respond to fire drills in their area and to assist visitors, volunteers, or physicians
who may not be accustomed to drills in this area. Personnel in the area of the fire’s point of
origin must follow the steps outlined in the R.A.C.E. procedures.

R = Remove all persons in immediate danger to safety. This includes patients, visitors, students,
or employees.
A = Activate manual pull station/dial 911.
C = Close all doors, windows to prevent the spread of smoke and fire.
E = Extinguish the fire.

• All staff are trained to use the pull stations located in the following areas.
• All staff are trained to use the extinguishers located in the following areas.
• The fire will need to be contained until the arrival of the fire department personnel. This
can be accomplished by closing doors . The fire can be suppressed using the fire
extinguishers located in the immediate area.
• Employees are also expected to use the P.A.S.S. procedure when extinguishing the fire.

P = Pull the pin on the extinguisher

A = Aim at the base of the fire
S = Squeeze the handles together
S = Sweep the fire from side to side

• All staff knows the locations of oxygen and gas supply cut off valves, which are located
______________________________________________________________.

2. The use and function of the fire alarm system and life safety system

There are many types of alarms utilized in the Medial Center buildings. Manual pull stations,
smoke detectors, heat detectors and water-flow indicators are some examples of these.
Activation of any of these alarms in any part of the building sends a signal to
_________________________________.
All alarms transmitted to are then transmitted to __________________________________ are
 Section 3 Fire Safety
Chapter 6
Fire Drills, Site-specific Fire Plan, and References

then transmitted to
_______________________________________________________________________ .

Employees should know the basic function of the following fire alarm components:

Smoke detectors — A device that senses visible or invisible particles of combustion (smoke).

Heat detectors — A device that senses a change in temperature above a fixed setting (usually
135
degrees).

Manual pull station — A device that personnel use to activate the fire alarm/life safety system.

Sprinkler tamper switch — A device used to detect that the water supply has been closed.

Water-flow alarm — A device used to detect any flow of water from a sprinkler system.

Sprinkler system — A suppression system employing automatic sprinklers attached to a piping

system containing water and connected to a water supply so that water discharges immediately
from sprinklers opened by heat from a fire.

Smoke damper — An automatically closing device used to stop the movement of smoke
through a duct penetration of a required fire wall or smoke wall.

Duct detectors — A smoke detector located in ducts for the purpose of fan shutdown in the
presence
of smoke.

Activation of the building fire alarm will require the immediate evacuation of the facility.

3. The roles and responsibilities in preparing for building evacuation.

Patients that are unable to walk will be transported by wheelchair and /or beds. The
ambulatory patients will be led as a group to a safe area (area of refuge).

Specific duties of personnel in the department:

• All staff will use the R.A.C.E. to evacuate patients and themselves.
• All personnel will know all exits that can be used for evacuation. Evacuation plans
should be consulted to pre-determine the location of these exits. The best route will
have to be determined at the time, considering the location of the fire.
• Medically and physically challenged persons should be moved by the nursing staff in
nursing areas. Medically and physically challenged employees, in non-nursing areas,
 Section 3 Fire Safety
Chapter 6
Fire Drills, Site-specific Fire Plan, and References

should be covered under the Duke University Policy for Medically and Physically
Challenged Individuals. Currently there are/are no Physically or Medically challenged
individuals employed at this location. (In the event someone were hired that meets the
criteria of this policy it would be consulted and a specific policy for that individual
implemented.)

The predetermined area(s) of refuge for this area is

___________________________________________.

The procedure for moving medically and physically challenged employees is

______________________________________________________________________________
_______.
DO NOT USE ELEVATORS FOR EVACUATION!

4. The location and proper use of equipment for transporting patients to the area of refuge
and for building evacuation. (Not needed in non-patient care area)

• Wheelchairs located in the front lobby (2) and the Pediatric Clinic(1).
• If life support or emergency transport is needed the Durham EMS will be called at 911.

5. The procedures personnel must follow to contain smoke and fire through building
compartmentalization procedures:

Fire walls and fire doors are designed and constructed with a specific fire resistance rating to
limit the spread of fire and restrict the spread of smoke. Smoke walls and smoke doors are
designed and constructed to restrict the movement of smoke. Smoke walls and smoke doors
may or may not have a fire resistance rating. Doors are instrumental in the containment of
smoke or fire. ALL doors must be closed and any equipment in the corridors will be moved.

_______________________________ Dept. Mgr./Dir. ________________ Approval by Safety

Committee

REFERENCES
Fire Protection; 29 CFR 1910 Subpart L
North Carolina Building Code, Vol V, Fire Prevention; 1998
National Fire Protection Association (NFPA) Standards
 Section 4 Use of Laboratory Animals
Chapter 1
Introduction and Purpose

Laboratory Safety Manual

Section 4
Use of Laboratory Animals

INTRODUCITON

PURPOSE

Proper care and use of laboratory animals is not only the humane thing to do, it is the law.
Together, Duke’s Institutional Animal Care and Use Committee (IACUC) and Division of
Laboratory Animal Resources (DLAR), maintain a program which assures compliance with the
Animal Welfare Regulations and the Public Health Service Policy on Humane Care and Use of
Laboratory Animals. For information about regulatory compliance or to request a copy of the
Duke University Manual for Animal Research, contact the DLAR at 919-684-2797.

Employees working with animals in a research environment may be exposed to a variety of

occupational risks including animal bites and scratches, needle sticks, back injuries from lifting
large items, exposure to zoonotic agents, allergens, or hazardous chemicals, etc. In order to
prevent accidents or exposures from occurring, all employees must understand the risks
involved with such work, and must also be familiar with basic safety precautions for work with
animals.

All proposed work involving animals must first be reviewed and approved by Duke’s IACUC
before work can begin. The IACUC's homepage is found at: http://vetmed.duhs.duke.edu/

All orders for vertebrate animals must be placed by the DLAR. Purchase orders will be accepted
only if accompanied by an approved Animal Use Protocol number.
 Section 4 Use of Laboratory Animals
Chapter 2
EOHW and Animal Handler Placement Reviews

Employee Occupational Health and Wellness (EOHW) and Animal Handler Placement Reviews

All employees who work with animals are required to complete the Placement Health Review
for Animal Handlers. This form will be reviewed by Employee Occupational Health and
Wellness, which administers all applicable medical surveillance. For more information, contact
EOHW at 919-684-3136.
 Section 4 Use of Laboratory Animals 
Chapter 3 
Personnel Training 

Personnel Training  

Duke’s IACUC provides animal care and use training sessions which are mandatory for all animal 
handlers.  The two required sessions are available online.  These trainings provide detailed 
instruction on both regulatory compliance and basic husbandry. Because hazards vary from lab 
to lab, it is also necessary for each principal investigator (PI) to provide site‐specific instructions 
for conducting animal work safely. This would include information on any etiologic agents, 
chemical hazards, radiation hazards, behavioral concerns of a particular animal species, etc. In 
addition to identifying the risks, the PI is also responsible for providing appropriate personal 
protective equipment such as lab coats, disposable gloves, goggles and face shields.  

 
 Section 4 Use of Laboratory Animals 
Chapter 4 
Husbandry 
Husbandry (DLAR website) 

Animals shall be provided nutritionally adequate food, potable, non‐contaminated water and a 
sanitary environment in which the animal’s health shall not be affected.  

 
 Section 4 Use of Laboratory Animals
Chapter 5
Injections
Injections

All sharps shall be disposed of immediately after use in an approved sharps container. Needles
shall not be broken, bent, or recapped before disposal. A one-handed recapping procedure may
be incorporated only after approval by the OESO-Biological Safety Division 919-684-8822.
Physical restraint procedures should be developed and practiced to prevent accidental
autoinoculation while at the same time reducing stress on the animal.
 Section 4 Use of Laboratory Animals 
Chapter 6 
Animal Restraint 
Animal Restraint  
   
Proper restraint and handling techniques are essential for reducing stress to laboratory animals, 
while at the same time allow animal care workers to perform their work with less chance of 
being scratched, bitten, kicked, etc. Animals can be restrained either manually or with restraint 
devices. It is the responsibility of the PI to train their staff on proper restraint for each species 
used.  

 
 Section 4 Use of Laboratory Animals 
Chapter 7 
Changing Bedding 
Changing Bedding  

Precautions should be taken, while changing animal bedding, to minimize or eliminate the 
aerosolization of hazardous agents which may have been shed by the animal. The use of a 
biological safety cabinet (BSC) or chemical fume hood should be used when changing animals 
dosed with hazardous agents. Many allergens can also be aerosolized during bedding changing. 
Some options include the use of a cage changing station, or decontaminating the soiled bedding 
before disposal by placing the whole cage in a biohazard bag and then autoclaving it. Cage 
changing stations are not appropriate for animals dosed with biohazardous material and 
autoclaving may not be appropriate for those dosed with chemical toxins. Please read and 
adhere to the procedures in the written standard operating procedures (SOP) for handling 
animals dosed with these hazardous materials. 

 
 Section 4 Use of Laboratory Animals
Chapter 8
Allergens
Allergens

One of the most common conditions that affect individuals who work with laboratory animals is
allergies. Typically, allergies to animals result from repeated exposure to an animal’s dander,
urine, saliva, serum, or other body tissues. Symptoms can range from mild (i.e. itchy or runny
nose and eyes) to severe (i.e. shortness of breath or red, itchy wheals on skin).

Levels of airborne allergens tend to rise significantly with certain activities such as changing or
cleaning animal cages. The use of ventilated hoods (cage changing station, BSC, or chemical
fume hood) for cage changing, dust-free bedding, or filtered caging systems, are all good ways
of reducing the level of airborne allergens. If these options aren’t available or feasible for a
particular situation, then personal respiratory protection may be warranted. Make an
appointment with Employee Occupational Health and Wellness (919-684-3136) or Student
Health (919-681-WELL) if you have allergy concerns. Contact Occupational Hygiene and Safety
(919-684-5996) for more information about respirators.
 Section 4 Use of Laboratory Animals
Chapter 9
Carcass Disposal
Carcass Disposal

All non-radioactive animal carcasses are to be collected by the DLAR staff and incinerated. For
more information, contact the DLAR at 919-684-5567.

Radioactive carcasses and their associated lab waste (i.e. bedding, excreta, sharps, etc.) are to
be bagged and sealed in 3 mil plastic bags. Make sure that all sharps are contained in a
puncture resistant container before placing in plastic bags! Once properly barcoded, the bags
are refrigerated in a lined 30 gallon plastic drum. All packaging and labeling materials are
provided by the OESO. For more information contact the OESO- Environmental Programs
Division at 919-684-2794.
 Section 4 Use of Laboratory Animals
Chapter 10
Working with Hazardous Materials
Working with Hazardous Materials

Standard operating procedures (SOP) must be developed and approved by OESO for any work
which involves the use of hazardous materials in animals. Such procedures shall detail the safe
handling of the animal throughout the duration of exposure.

• For work with biological agents (i.e. viruses, bacteria) in animals, see the Guide for
Developing an SOP for the Use of Biohazards in Animals. Contact Biological Safety
Division (919-684-8822) for assistance.
• For work with hazardous drugs or other toxic chemicals in animals, see the SOP for
Work with Toxic Chemicals in Animals. Contact Occupational Hygiene and Safety (919-
684-5996) for assistance.
• For work with radioactive materials in animals, contact the Radiation Safety Division at
919-684-2194 for questions about SOP development.
 Section 4 Use of Laboratory Animals 
Chapter 11 
Animal Biosafety Levels 
Animal Biosafety Levels 

Work involving the exposure of animals with biological materials must be conducted at the 
appropriate containment level to ensure adequate protection of personnel and the 
environment.  The following table summarizes the Center for Disease Control and Prevention's 
four animal biosafety levels. 

ABSL   Agents   Practices   Safety Equipment    Facilities  

(Primary Barriers)   (Secondary Barriers) 
1  Not known to cause  Standard animal care and  As required for care of  Standard animal facility 
disease in  management practices,  each species   non‐recirculation 
immunocompetent   including appropriate  of exhaust air  
adult humans  medical surveillance   directional air 
programs  flow 
recommended  
2  Associated with  ABSL‐1 practices plus:  ABSL‐1 equipment  ABSL‐1 facility plus: 
human disease.     primary barriers:  
Hazard:   limited access   containment   autoclave 
autoinoculation,   biohazard warning  equipment  available  
ingestion, mucous  signs   appropriate for   handwashing sink 
membrane exposure   sharp precautions species: PPE:  available in the 
 biosafety manual  laboratory coats,  animal room  
 decontamination  gloves, face and 
of all infectious  respiratory 
wastes and of  protection as 
animal cages prior  needed 
to washing  
3  Indigenous or exotic  ABSL‐2 practices plus: ABSL‐2 equipment plus: ABSL‐2 facility plus: 
agents with potential   
for aerosol   controlled access   containment   physical 
transmission; disease   decontamination  equipment for  separation from 
may have serious or  of clothing before  housing animals  access corridors  
lethal consequences  laundering   and cage   self‐closing, 
 cages  dumping  double door 
decontaminated  activities   access  
before bedding   Class I or II   sealed 
removed   biosafety  penetrators  
 disinfectant foot  cabinets available   sealed windows  
bath as needed   for manipulative   autoclave 
procedures  available in 
(inoculation,  facility  
necropsy) that 
may create 
infectious 
aerosols. PPE: 
appropriate 
respiratory 
protection  
 Section 4 Use of Laboratory Animals 
Chapter 11 
Animal Biosafety Levels 
ABSL  Agents  Practices   Safety Equipment    Facilities  
(Primary Barriers)   (Secondary Barriers) 
4  Dangerous/exotic  ABSL‐3 practices plus: ABSL‐3 equipment plus:  ABSL‐3 facility plus: 
agents which pose 
high risk of life‐  entrances through   maximun   separate building 
threatening disease,  change room  containment  or isolated zone  
aerosol‐transmitted  where personal  equipment   dedicated 
lab infections; or  clothing is  (i.e.Class III  supply/exhaust, 
related agents with  removed and  biosafety cabinet  vacuum and 
unknown risk of  laboratory  or partial  decontamination 
transmission  clothing is put on;  containment  systems  
shower on exiting  equipment in   other 
 all wastes are  combination with  requirements 
decontaminated  full body, air  outlined in the 
before removal  supplied positive‐ complete 
from facility   pressure  description of 
personnel suit)  ABL‐4  
used for all 
procedures and 
activities  

Summarized from Biosafety in Microbiological and Biomedical Laboratories, 5th Edition, 2007.  

  

 
 Section 4 Use of Laboratory Animals
Chapter 12
Safe Use of Anesthetic Gases and References
Safe Use of Anesthetic Gases

Many anesthetic gases are used at Duke University for performing animal surgeries. These
gases can present a risk for potential exposure to the lab personnel performing the surgeries.
Anesthetics of concern include ether, nitrous oxide, and halogenated agents (i.e. halothane,
isoflurane, methoxyflurane). Some of these halogenated anesthetics have been linked to
adverse health effects in exposed workers, such as reproductive and neurological effects.

Emphasis must be placed on protecting personnel from exposure by adequately “capturing” the
waste gas being generated. This may be accomplished by several methods depending on the
method of delivery of the gas.

The following good-practice should be considered:

For enclosed chambers (i.e. Bell jar):

· perform work in a fume hood so when lid is removed, gases are captured by hood

· evacuate chamber via building vacuum system prior to removing lid

· make sure the chamber lid is tight-fitting

· remove chamber lid only when animal is being placed into or removed

For anesthetic gas machines:

· verify that proper filtration canisters are installed

· filter canisters must be maintained according to manufacturer’s specifications

· choose appropriate sized face-piece to ensure most efficient waste gas recovery

Lab personnel that are concerned with possible exposure to anesthetic gases may contact the
Occupational Hygiene & Safety division of OESO at 919-684-5996 to request an exposure risk
evaluation.

REFERENCES
Guide for the Care and Use of Laboratory Animals; NRC, 1996
Guidelines for Research Involving Recombinant DNA; NIH, 2009
Biosafety in Microbiological and Biomedical Laboratories, 5th Ed.; CDC/NIH, 2007
 Section 5 Laboratory Start‐up and Closeout 
Chapter 1 
Introduction and Purpose 

Laboratory Safety Manual  
Section 5 
Laboratory Start‐up and Closeout 

                                                                                                                                     
INTRODUCTION                                                                                                                                                    
PURPOSE 

This section has been included to provide guidance to all principal investigators (PIs) and 
laboratory managers on appropriate “start‐up” and “close‐out” procedures. It is imperative that 
these procedures be followed to ensure compliance with all applicable federal, state and local 
requirements.  

 
 Section 5 Laboratory Start-up and Closeout
Chapter 2
Instructions for Principle Investigators

Instructions for Principle Investigators (PI)

Background

It is necessary that each PI be made aware of all applicable safety requirements. Failure to
incorporate required work practices may lead to an unsafe occupational setting. Such non-
compliance may also result in fines from external regulatory agencies such as the Occupational
Safety and Health Administration (OSHA).

Procedures

New PIs are to complete the “Notice of Laboratory Occupancy” form and fax to the OESO at
919-681-7509 (prior to arrival if possible). Once notice is received, an OESO representative will
contact the PI to schedule an on-site visit, at which time, all applicable safety policies and
procedures will be discussed. Every effort will be made to schedule the on-site visit within the
first week of occupancy. Thereafter, laboratory safety audits will be conducted by the OESO on
a periodic basis (at a minimum within one month of the annual due date).

The following special requirements may be applicable to your laboratory operations. See the
links for more information and/or call OESO-Laboratory Safety Program 919-684-8822 for
guidance.

• Lab-specific Chemical Hygiene Plan (required of all labs who use chemicals): Each
laboratory must have a Laboratory-Specific Chemical Hygiene Plan, list of Particularly
Hazardous Substances (PHSs) used in the lab, and lab-specific Standard Operating
Procedures (SOPs) for any PHSs and nanomaterials used in the lab.

• Chemical Waste Disposal: All new PIs must carefully review the Duke Chemical Waste
Policy. If any chemical waste will be produced, information on proper disposal can be
found at: http://www.safety.duke.edu/EnvPrograms/ChemWasteGen.htm.

• Application for Possession of Radioactive Materials: An application must be submitted

to the Radiation Safety Officer (RSO) for the use of all radioactive materials. Only after
the application has been reviewed and approved by the RSO, shall the laboratory be
allowed to purchase or use radioactive materials. Information on how to become an
“authorized user” can be found at: http://www.safety.duke.edu/RadSafety/new_au.asp. Contact
Radiation Safety Division 919-684-2194 for more information.

• Registration for Work with Recombinant DNA: Experiments involving the utilization of
rDNA may require approval by the Duke University Institutional Biosafety Committee
(IBC) prior to submission to outside agencies and the initiation of experimentation. PIs
should refer to the rDNA chapter of the Biological Safety Section of this manual and/or
 Section 5 Laboratory Start-up and Closeout
Chapter 2
Instructions for Principle Investigators

contact OESO - Biological Safety Division 919-684-8822 for more information. To go

directly to the rDNA project registration form click here.

• Written standard operating procedures (SOP) for work with biohazards and/or
hazardous chemicals:
o Biosafety Level 2 (BSL2) SOP:
http://www.safety.duke.edu/LabSafety/Docs/Combined%20ABSL-2%20and%20BSL-
2%20SOP%20Template.doc.
o Hazardous Chemical SOP: http://www.safety.duke.edu/OHS/chemsopsTemplates.htm

• Personal Protective Equipment Hazard Assessment:

http://www.safety.duke.edu/OHS/Documents/PPE%20Hazard%20Assessment%20Form.pdf
 Section 5 Laboratory Start‐up and Closeout 
Chapter 3 
Laboratory Closeout and Appendices 
 
Laboratory Closeout  

Procedures  

It is imperative that all laboratory closeouts be conducted while conforming to standard 
procedures for the removal of hazardous materials. The OESO should be notified at least 30 
days prior to the anticipated departure date. Notice is given by completing the Laboratory 
Closeout Notice and forwarding it to the Lab Closeout Coordinator (information is on the form). 
If the lab is simply moving to another Duke Laboratory building, the Laboratory Relocation 
Notice should be completed.  Upon receipt, the OESO will provide specific instructions for 
proper shut‐down to the laboratory’s assigned safety contact. The departing principal 
investigator shall be held fully responsible for all Institutional requirements. The laboratory will 
be cleared for new occupancy only after all requirements are met.  

Should proper notification not be given, the principal investigator and/or the department will 
be held responsible for all cost incurred for safe disposal of remaining hazardous material 
wastes.  

The following is a list of requirements which must be met for each class of hazardous agents 
used before a laboratory is released by the OESO.  

Biological Hazards  

1. All biological materials (i.e. blood, fresh tissue, bacterial cultures, etc.,) must be removed 
from the laboratory by disposing according to Institutional policy, by shipping to another facility 
while conforming to the approved shipping regulations, or by transferring to another PI. This 
includes those materials stored in refrigerators, freezers, incubators and cold rooms.  

2. All equipment which has come in contact with potentially infectious materials must be 
properly decontaminated and labeled with the “Laboratory Equipment Statement of Hazard 
Assessment”. 

3. All biological waste must be properly decontaminated and disposed of appropriately 
(autoclave, etc.).  

4. All benchtops or other work surfaces on which biological materials were manipulated must 
be wiped down with an approved disinfectant.  

5. The OESO shall determine the appropriate decontamination method for all biological safety 
cabinets. If formaldehyde gas decontamination is deemed necessary, the departing PI will be 
financially responsible.  
 Section 5 Laboratory Start‐up and Closeout 
Chapter 3 
Laboratory Closeout and Appendices 
 
Chemical Hazards  

1. All chemical containers are labeled with the chemical name or a best description of the 
compound.  

2. All chemicals not transferred to another Duke laboratory have been submitted as chemical 
waste using the on‐line “Waste Pickup Request System”.  

3. Chemicals being shipped or transferred to another facility must be packaged and labeled 
according to approved regulations.  

4. Compressed gas cylinders are to be returned to their supplier (e.g., National Welders). 
Cylinders owned by the PI (i.e., lecture cylinders) may be submitted to the OESO‐Environmental 
Programs for proper disposal.  

Radiological Material Hazards  

1. Notify the Radiation Safety Officer of intention to terminate authorization.  

2. Dispose of all radioactive materials by one of the following methods:  

 Transfer materials to another authorized user while complying with all license 
restrictions of that user.  
 Disposal of materials through the OESO‐Environmental Programs Division. Shipment of 
materials to a non‐Duke licensee while conforming to approved shipping regulations.  

Note: There will be notification/acceptance requirements at the new facility.  
 
3. Perform a thorough radiation contamination survey of the laboratory, including equipment 
to determine if allowable contamination levels are achieved. Those areas found to exceed the 
allowable limits must be decontaminated and resurveyed until within allowable limits.  
   
   
APPENDICES 
Laboratory Startup/Closeout/Relocation Notices 
Laboratory Safety Audit Checklists 
 
 
 Laboratory Safety Manual
Important Numbers

Emergency Numbers Regulated Laboratory Waste

• Medical Emergency: 911 • General Concerns: 919-684-8822
• Duke University Police: 911 from a • Biological Waste (Sharps Boxes/BFI
campus phone; 919-684-2444 Disposal): 919-681-2727
from a cell phone • Chemical Waste: 919-684-2794
• Chemical Spill: 911 from a campus • Radioactive Waste: 919-684-2794
phone; 919-684-2444 from a cell
phone Industrial Hygiene
• Radioactive Materials Spill: 911 • General Concerns: 919-684-5996
from a campus phone; 919-684- • Asbestos Abatement: 919-684-5996
2444 from a cell phone • Noise/Air Monitoring: 919-684-5996
• Blood/Body Fluid Exposure: 115 • Reproductive Health: 919-684-5996
(on campus), 919-684-8115 (off • Respiratory Protection: 919-684-
campus) 5996
• To report workplace safety
concerns/ unsafe working Other
conditions : 919-684-2794 • Employee Occupational Health and
• To report odors : 919-684-2794 Wellness (EOHW) 919-684-3136
• Environmental Services Uniform
Safety Shop (Lab Coat Laundering): 919-
• Occupational & Environmental 684-3784
Safety: 919-684-2794 • Division of Laboratory Animal
• Contractor/Work-Site Safety: Resources: 919-684-2797
919-684-5996 • Grants & Contracts: 919-684-3030
• Clinical/Research Laboratory • University Maintenance 919-684-
Safety: 919-684-8822 2122
• Fire Safety: 919-684-5609 • Medical Center (buildings)
• Radiation Safety: 919-684-2194 Maintenance 919-684-3232
• Ergonomics: 919-668-(ERGO)3746

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