Principles & Concepts

Principles & Concepts

The purpose of this section is to provide definitions of biosafety concepts.

Biohazardous Material

Biohazardous materials are any microorganism, or infectious substance, or any naturally occurring, bioengineered, or synthesized component of any such microorganism or infectious substance, capable of causing:

1. Death, disease, or other biological malfunction in a human, an animal, a plant, or another living organism
2. Deterioration of food, water, equipment, supplies, or material of any kind
3. Harmful alteration of the environment.

These include, but are not limited to:

• Certain bacteria, fungi, viruses, rickettsiae, protozoa, parasites
• Recombinant products
• Listed Select Agenda and Toxins (exempt and non-exempt quantities)
• Allergens
• Cultured human or animal cells and the potentially infectious agents these cells may contain
• Viroids and prions
• Other infectious agents as outlined in laws, regulations, or guidelines.

Examples include:

• All materials containing rDNA
• Transgenic Animals or Plants
• Human, Animal or Plant pathogens
• Biological toxins (such as T-2 toxin, tetrodotoxin, etc.)
• Human blood and certain human body fluids
• Select agents
• High consequence livestock pathogens and toxins
• Human or monkey cell cultures

Specifically excluded from this definition is all non-rDNA BSL-1 research activities.

Pathogenicity or Virulence

Pathogenicity or virulence is the ability of a biohazardous material to produce or develop a rapid, severe, or deadly disease. Some materials are highly pathogenic, even in healthy adults, whereas others are opportunistic pathogens able to infect only hosts with lowered immunity or sites other than their normal habitat. Some biohazardous materials are attenuated, or weakened, and do not produce significant disease. The more severe the potentially acquired disease, the higher the risks.

Routes of Entry

An infection occurs when pathogenic microorganisms enter the human body in sufficient numbers and by a particular route which overcomes the body’s defense system. By understanding the mode of transmission (pathway from source to you) and route of entry (entry route into body), procedures or controls to prevent exposure and infection can be developed.

Inhalation hazards: Inhalation of aerosolized biohazardous materials is the most common route of entry into the body. Inhalation of aerosols involves microscopic solid or liquid particles small enough to remain dispersed and suspended in air for long periods of time. Sources of aerosols include:

• Aerosolized solid material (spores, dust, particulate, etc.).
• Liquid material (mists and sprays, coughing, spittle, sputum, etc.).
• Technical process (blending, grinding, sonicating, lyophilizing, sawing, centrifuging, etc).

Ingestion hazards: Ingestion of biohazardous materials occurs frequently as the result of poor personal hygiene and poor laboratory practice. Proper hand washing minimizes the opportunity for mouth and eye exposures. Examples of how ingestion occurs include:

• Eating, drinking, and smoking in laboratory
• Mouth pipetting and suction techniques
• Transfer of microbes to mouth by contaminated fingers or articles

Direct (Skin/Eye) Contact hazards: Direct contact to biohazardous materials occurs through cross-contamination and mucous membrane exposure including the skin, eyes, inside of the mouth, nose, and the genitals. The main avenues by which biohazardous materials enter the body through the skin are hair follicles, sebaceous glands, sweat glands, and cuts or abrasions. Examples of how ingestion occurs include:

• Splash or spray of biohazardous material onto skin, eye, mouth, or nose
• Handling contaminated equipment with unprotected non-intact skin
• Transfer or rubbing by contaminated fingers or gloved hand
• Applying cosmetics or contact lens in laboratory

Injection or inoculation hazards: Inoculation or injection occurs when biohazardous material is accidentally introduced into the body with contaminated objects through the intact skin barrier. Inadequate control of sharp instruments and infected animals or arthropod vectors usually results in accidental inoculation or injection. Examples of injection and inoculation hazards include:

• Inoculation with a hypodermic needle, broken glassware, scalpels, or other sharp instruments
• Sharps injuries (needle sticks, glass pipettes, syringes, etc.)
• Animal bites, scratches, kicks, abrasions, punctures

Agent Stability or Viability

Stability and viability refer to the ability of a biohazardous material to retain its biohazardous characteristics such as aerosol infectivity and survival time in environment. Factors such as temperature, humidity, pH, oxygen, sunlight or ultraviolet light, chemical disinfectants, growth factors (food reservoir or media), and competition with endemic organisms must be considered.

Infectious Dose

The infectious dose is the number of microorganisms required to initiate an infection. This dose can range from one to hundreds of thousands of units depending on agent, exposure route, virulence, and host immune status or susceptibility for the disease.

Concentration (Amount of Agent)

Concentration is the number of infectious organisms per unit volume. As the viable agent concentration and volume increases, the risk potential gets higher. The media/reservoir, laboratory activity, volume (especially >10 liters) need to be considered in risk determination.

Immune Status

Immune status is the current condition of a living organism to resist and overcome infection or disease. The primary function of the immune system is to protect the body from foreign substances by an acquired ability to distinguish self from non-self. Host susceptibility or immune status helps determine the level of risk of acquiring a disease upon exposure. CDC and NIH guidelines presume a population of immuno-competent individuals.

Laboratory Biosafety Level Criteria

There are four recommended laboratory biosafety levels. The biosafety levels consist of laboratory practices, safety equipment, and facilities combinations which are specifically appropriate for the operations performed, suspected routes of biohazardous material transmission, and laboratory function or activity.

Biosafety Level 1 (BSL-1)

• Suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment.
• Special containment equipment or facility design is neither required nor generally used.
• Laboratory personnel have specific training in the procedures conducted in the laboratory.
• Supervision by a scientist with general training in microbiology or a related science.

Biosafety Level 2 (BSL-2)

• Suitable for work involving agents of moderate potential hazard to laboratory personnel and the environment.
• Laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists.
• Access to the laboratory is limited when work is being conducted.
• Extreme precautions are taken with contaminated sharp items.
• Certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment.

Biosafety Level 3 (BSL-3)

• Clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route.
• All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment.
• The laboratory has specific engineering and design features.
• Laboratory personnel have specific training in handling pathogenic and potentially lethal agents.
• Supervision by a competent scientist who is experienced in working with these agents.

Biosafety Level 4 (BSL-4)

• Work with dangerous and exotic agents that pose a high individual risk of aerosol transmitted laboratory infections and life threatening disease.

Summary Table

Vertebrate Animal Biosafety Level Criteria

There are four recommended vertebrate animal biosafety levels. The recommendations below describe practices, safety equipment and facilities for experiments with animals infected with agents that cause, or may cause, human infection. In general, the biosafety level recommended for working with biohazardous material in vivo and in vitro are comparable.

Animal Biosafety Level 1 (ABSL-1)

• Suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to personnel handling the animals and the environment.

Animal Biosafety Level 2 (ABSL-2)

• This level is involves practices for work with those agents associated with human disease. It addresses hazards from ingestion as well as from percutaneous and mucous membrane exposure. ABLS-2 builds upon the practices, procedures, containment equipment, and facility requirements of ABLS-1.
• ABLS-2 is suitable for work involving agents of moderate potential hazard to laboratory personnel, animals, and the environment.
• Laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists.
• Access to the animal facility is limited to the fewest number of individuals possible. Personnel who must enter the room for program or service purposes when work is in progress are advised of the potential hazard.
• Certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment.

 Animal Biosafety Level 3 ABSL-3

• ABLS-3 level involves practices suitable for work with animals infected with indigenous or exotic agents that present the potential of aerosol transmission and of causing serious or potentially lethal disease. ABSL-3 builds upon the standard practices, procedures, containment equipment, and facility requirements of ABSL-2.
• All procedures involving the manipulation of infected animal and infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment.
• The animal facility has specific engineering and design features.
• Laboratory personnel have specific training in handling infected animals with pathogenic and potentially lethal agents.

Animal Biosafety Level 4 (ABSL-4)

• This level involves practices suitable for addressing dangerous or exotic agents that pose high risk of life threatening disease, aerosol transmission, or related agents with unknown risk of transmission.

Summary Table

Recombinant DNA Biosafety Level Criteria

The following are NIH requirements for Campus Research with Recombinant DNA in humans, animals, and plants. The investigator must make an initial risk assessment based on the Risk Group (RG) of an agent. Agents are classified into four Risk Groups (RGs) according to their relative pathogenicity for healthy adult humans by the following criteria:

Risk Group 1 (RG1): agents are not associated with disease in healthy adults.

Risk Group 2 (RG2): agents are associated with human disease that is rarely serious and for which preventive or therapeutic interventions are often available.

Risk Group 3 (RG3): agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available.

Risk Group 4 (RG4): agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available.

NIH Guidelines also address physical and biological containment for Recombinant DNA research involving humans, animals, plants and large scale use, including standard microbiological practices, special practices, containment equipment and laboratory facilities.

• Biosafety Level 1-4 (BL1 to BL4): standard research laboratory experiments.
• Biosafety Level 1-4 Large Scale (BL1-Large Scale to BL4-Large Scale): large scale (over 10 liters) research and production with Good Large Scale Practices (GLSP).
• Biosafety Level 1-4 Plants (BL1-P to BL4-P): standard plant greenhouse facility experiments.
• Biosafety Level 1-4 Animals (BL1-N to BL4-N): standard whole animal facility experiments.