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TN_1_061205.doc

D O C U M E N T

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TECHNICAL NOTE TN1:

RESULTS OF LITERATURE REVIEW

Peter Mani Andrea Friedli

prepared by/préparé par P. Mani reference/réference COR-PP-SOW-HME-0104

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date of issue/date d’édition 31-12-2006

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Containment Technology Research Study: TN1 SoW ref. COR-PP-SOW-HME-0104

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A P P R O V A L

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Peter Mani, Andrea Friedli date

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31-12-2006

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C O N T E N T S

Review of International and National Biosafety Standards Introduction Procedure of Literature Analysis Overview of Biosafety Level 4 Standards

- Occupational Safety - Equipment Requirements - Primary Containment - Secondary Containment - Tertiary Containment

Conclusions Bibliography

A C R O N Y M S

BMBL Biosafety in Microbiological and Biomedical Laboratories

BSC Biological Safety Cabinet

BSL Biosafety Level

CDC Centres for Disease Control and Prevention (USA)

FLI Friedrich-Loeffler-Institut

GMO Genetically Manipulated Organisms

HEPA High Efficiency-Particulate Air

HVAC heating, ventilating, and air conditioning

IATA International Air Transport Association of Dangerous Goods

MCL Maximum Containment Laboratory

NIH National Institutes of Health (USA)

PC Protection Containment

WHO World Health Organization

1

Review of International and National Biosafety Standards

Since the question of reasonable concrete requirements in the field of biosafety usu-

ally has been a national concern, it is a challenge to find in this «jungle» of biosafety

norms and regulations a consistent set of recommendations which could be useful on

an international level. Manuals and reports of international organisations like the WHO

do a first step in this direction, but often they are more or less in line with national

US standards. This overview additionally takes into account national regulations and

recommendations of European Countries (see below).

Most of the regulations are based on the biosafety classification which was developed

by the Centres for Disease Control and Prevention (CDC) and the National Institutes

of Health (NIH) of the U.S. Department of Health and Human Services. Today the clas-

sification of laboratories into four biosafety levels made on the basis of the potential

hazard of the agent and of the laboratory's function or activity is accepted world wide.

However, no binding regulations and standards concerning biosafety measures exist,

which are effectual and mandatory on an international level. And, as Holeman and

Gueltekin justifiably remark, “the properties of biological agents do not alter simply

because geographical boundaries or jurisdictional responsibilities change” [1]. Particu-

larly with ongoing internationalisation and globalisation the question of international

standards becomes more and more pressing.

Lacking internationally binding standards, as well as the fact that many new laboratories

all over the world are proliferating at the moment, result in a variety of different defini-

tions of the conditions, which have to be met by a laboratory in order to attain a certain

biosafety level. The most disputed are requirements for laboratories of Biosafety Level

4, which are also referred to as high containment laboratories. In the Anglophone areas

such laboratories are called BSL-4 (Biosafety Level 4) or MCL (Maximum Containment

Laboratory); only in Australia this level is described by PC4 (Protection Containment

Level 4). In France such labs are referred to as P4 (protéction 4) labs; in Germanophone

countries they are called "Biosicherheitsstufe 4".

Additionally there are different design concepts contributing to the confusing defini-

tions. Concerning work with animal pathogen agents, laboratories are still described as

Biosafety Level 4, even if the viruses are not dangerous for humans - this is to show,

2

that the environment and the animals are maximally protected. In the US those labs are

described as BSL-3 ag (ag is for agriculture). In a German project (FLI Riems), labora-

tories of this kind are called L4vet. Furthermore it is in principle possible to work with

viruses of risk group 4 in a laboratory of level 3, provided that this occurs in a biosafety

cabinet of class III (a so called glove box). So, an unconsistency in terminology and

defiition futher make it difficult to formulate a uniform set of standards.

In the USA the guidelines and standards of the CDC in Atlanta are authoritative. Fur-

ther the regulations of the IATA (International Air Transport Association of Dangerous

Goods) as well as of the US Department of Agriculture are recognised. As already

mentioned above, those US national standards often influence recommendations on

an international level.

In recognised works with practical biosafety recommendations one often finds ample

documentation about Good Laboratory Practice, staff training and operational proce-

dures, which form the core of safe and secure work with dangerous organisms. But

actually, the first basis for biosafety and security measures is founded by the laboratory

design before construction. The International Veterinary Biosafety Working Group (IVBW)

for the first time tried to unify experiences and know-how from all over the world in a

handbook on design and construction of veterinary containment facilities. Even if this

state-of-the-art is focused on veterinary facilities, many of the findings are applicable

on the broader field of biosafety.

The following overwiev over different standard definitions for Level-4 laboratories con-

centrates exclusively on structural elements, meaning elements that are relevant for

the laboratory design and construction. For the analysis all available regulations on

Biosafety Level 4 from the following sources have been collected and structured:

• US Code of Federal Regulations [2]

•. EU Council Directive [3]

• Swiss Ordinance on the Contained Use of Organisms [4]

• German GMO Guideline [5]

• WHO Laboratory Biosafety Manual [6]

3

• Health Canada: Laboratory Biosafety Guidelines [7]

• CDC/NIH BMBL Guidelines [8]

Procedure of literature analysis

Out of the analysed documents a total of about 230 requirements were found. Identi-

cal or similar requirements were combined and structured in the three categories:

primary, secondary and tertiary containment. All three categories have been further

substructured in technical systems like HVAC, sewage treatment and so on (see fig.

1).

Figure 1: Raw data exerpted from the literature 2-8.

Finally, a synthesis was made by the remaining combined requirements in order to

4

have a common set containing more or less all relevant requirements of the original

set of 230 (see fig. 2). Few requirements have been discarded as they were found

too specific to one particular regulation and no equivalent could be found elsewhere.

The requirement for a double HEPA filtering of supply air in the German Guideline may

serve as an example for which no reason could be found and it may even be a typing

error. The resulting final set contains a total of about 70 specific requirements.

Fig. 2: Synthesis of the essentials

5

Overview of Biosafety Level 4 Standards

As a general remark it is to say that all BSL-4 activities are conducted in closed, gastight

Class III biological safety cabinets (glove-boxes) or in Class II BSCs in conjunction with

a one-piece positive pressure personal suit ventilated by a life-support system.

Occupational Safety

It is not allowed for a person to work alone unless there is a continuous intervisibility

by an observation window or a video monitor.

A communication for routine and emergency contacts must be established.

Visual or audible alarms have to be provided inside and outside the BSL-4 to signal air

handling and breathing air systems failure.

A competent person must be available outside the laboratory to assist in case of an

emergency.

Life safety systems, lighting, HVAC systems, biosafety cabinets, security systems and

other essential equipment has to be supported with emergency back-up power.

Interlocked doors, if present, have to have manual overrides for emergency exit.

The exhaust as well as the supply air systems have to be independent of other labora-

tory areas.

Scientific and technical rational of the standards is given in the

box beneath the concerning standard if necessary.

The focus of this overview is structural design and not organisational

principles. Therefore, there might be additional occupational safety

regulations outside design issues.

6

Equipment Requirements

A laboratory has to contain its own equipment.

Centrifuges for human pathogen organisms that are handled with in a glove-box labora-

tory can only be operated in adequate glove-boxes or in adequate rooms.

Primary Containment

Decontamination Measures

Bench tops must not have open seams; benches, doors, drawers, door handles etc.

have to have rounded rims and corners.

Handwashing sinks have to be provided with "handsfree" capability.

Safety and Security Measures

Internal facility appurtenances (e.g. light fixtures, air ducts and utility pipes) are ar-

ranged to minimize the horizontal surface area on which dust can settle.

A laboratory with Class III biosafety cabinets is only accessible through a minimum of

two doors.

Ventilation System

General:

The ventilation system must not recirculate exhaust air.

The production and release of aerosols can already be reduced by

using the appropriate equipment in a proper way. The laboratory de-

sign must include such principles.

7

Airflow control devices and duct sensors have to be located downstream of the exhaust

HEPA filter and upstream of the supply bubble tight backdraft damper or HEPA filter,

or if located upstream, duct penetrations have to be sealed.

Room pressure differential monitoring lines penetrating the containment barrier have

to be provided with filters of efficiency equal to that of HEPA filtration.

Suit laboratory:

Personnel who enter the suit area are required to don a one-piece, positively pressu-

Concerning the ventilation system, there exist several regulations referring to the category of primary containment. The recirculation of exhaust air is not allowed in BSL-4 laboratories in order to be sure that no aerosols could re-enter the ventilations cycle. Actually this seems to be paradox, since HEPA filters are considered not to let pass through an inacceptable amount of aerosols. It could be argu-mented that with the recirculation costs could be saved as supply air has to be pretreated, on the other hand a certain amount of the energy can be recuperated by heat recovery, Finally,according to statistics out of an amount of 1015 aerosols, 103 are passing through, which leads to the standard of a non-recirculated ventilation system in BSL-4.

The exhaust HEPA filters are usually located outside the containment perimeter which means that a part of the exhaust air duct to the filter housing is on the outside of the containment barrier. Consequently every device (that is potentially not contaminated) penetrating the duct has to be installed in the part of the duct inside the containment perimeter or downstream after the HEPA filter in order to guarantee a tight containment barrier.

8

rized, HEPA-filered, supplied-air suit.

Compressed breathing air has to be provided to the positive-pressure personal protec-

tive equipment (i.e. for connection to the air hose of suits), equipped with breathing air

compressors and back-up cylinders (sufficient for 30 minutes per person).

Air hose connections have to be provided in all areas where suits are worn, including

chemical shower and suit change room.

The positioning of the supply and exhaust points should be such that the dead air space

in the suit room is minimized.

Glove-box laboratory:

The glove-boxes are provided with individual, HEPA-filtered supply and exhaust air ducts

(exhaust air double filtered). The exhaust air of the glove-boxes has to be fed outside

through an own duct. The supply air may be drawn from within the room through a HEPA

filter mounted on the glove-box or supplied directly through the supply air system. If the

Class III BSC is connected to the supply system, it is done in a manner that prevents

positive pressurization of the cabinet. The Class III BSC must be directly connected to

the exhaust system.

Ductwork that penetrates into the containment space needs several considerations. Tightness of the duct towards the barrier and loca-tion of the diffusers assist pressure decay requirements and allows proper convection patterns within the containment space. Many cal-culations using air change per hour are flawed if the duct / diffuser penetrations are not sufficiently separated to avoid air short-circui-ting. The pictures in appendix x show how CFD computational fluid dynamics can assist with diffuser design and placement. In the three pictures the supply air diffuser throw direction and penetrations were improved from the first to the last figure, thereby reducing the throw velocities to within acceptable limits within a large animal room.

9

There has to be a negative pressure of ISO Pa in the glove-box relative to the pressure

in the room.

It must be guaranteed, that there will be an alarm in case of a power breakdown.

Secondary Containment

Airlocks

A device to transfer large pieces of equipment and the like has to be provided.

Airlock entry ports for specimens, materials and animals must be provided. A dunk tank

or a pass-through box, which can be fumigated, has to be provided in order to transfer

goods that are heat sensitive.

As in a glove-box laboratory the containment barrier is given by the walls of the glove-box, the same standards of a room ventilation systems of a suit lab are applied. The room is considered to be non-contaminated, so supply air can be fed into the glove box from the inside of the laboratory ba a simple HEPA filter only. Since there exists the possibility of e.g. loss of an attached glove, the glove box has to be under-pressurized (see also standard below).

Large equipment which cannot be autoclaved can be transferred through a decontamination chamber where methods such as fogging, vaporisation or gaseous methods can be applied .

Non heat resistant equipment and material cannot be autoclaved with steam (such as e.g. microelectronics) and must be decontaminated by other means in order to be transferred. A possible alternative is fumiga-tion in a decontamination chamber.

10

Supplies and materials needed in the facility are brought in by way of the double-doored

autoclave, fumigation chamber, or airlock which is appropriately decontaminated after

use. After securing the outer doors, personnel within the facility retrieve materials by

opening the interior doors of the autoclave, fumigation chamber or airlock. These doors

are secured after materials are brought into the facility.

Biological materials to be removed from the Class III BSC or from the maximum contain-

ment laboratory in a viable or intact state must be transferred to a sealed nonbreak-

able primary container, enclosed in a nonbreakable sealed secondary container, and

removed from the facility through a disinfectant dunk tank, fumigation chamber, or an

airlock designed for this purpose.

A three-chamber airlock consisting of a changing and/or showering area (suit labo-

ratory: chemical shower) and interlocking doors has to be provided for entering the

working rooms of the laboratory. Personnel must shower each time they leave the

facility; they may use the airlock to enter or leave the laboratory without showering

only in emergency.

Barrier Requirements

All penetrations have to be sealed with nonshrinking sealant at the containment bar-

rier.

Autoclaves that open outside the wall of the containment barrier must be sealed to

the wall of the containment barrier.

Openings into the the glove-box room are minimized and are capable of being sealed

to facilitate decontamination.

Since the shower is an airlock it represents the barrier of the contain-ment. For this reason, it has to consist of three chambers connected by interlocked doors in order to prevent a simultaneous opening. An intensi-ve air ventilation removes all the aerosols created during the showering.

11

Surfaces have to be continuous and compatible with adjacent and overlapping materi-

als (i.e. to maintain adhesion and a continuous perimeter). Continuity of seal has to

be maintained between the floor and wall (a continuous cove floor finish up the wall

is recommended.)

All supply and exhaust liquid and gas services are protected by devices that prevent

backflow. Gas ducts have to be provided with HEPA filters, liquid ducts with germtight

filters. Domestic water branch piping serving laboratory areas have to be provided with

a backflow prevention and an isolation valve, to be located in close proximity to the

containment barrier.

Sewer vents and other service lines contain HEPA-filters and protection against ver-

min.

The laboratory must not be linked to a general vacuum system. A portable vacuum

pump has to be provided in the laboratory. The internal contamination of this pump

has to be minimized (e.g. HEPA filtration of vacuum line, use of disinfectant traps). If

there is a central vacuum system present, then it must not serve areas outside the

facility and it has to have an in-line HEPA filter placed as near as practicable to each

use point or service cock.

There must not be any window in the laboratory; if there are windows, they have to be

sealed tight, shatter-proof and not to be opened.

Interior surfaces have to minimize movements of gases and liquid through perimeter

membrane. Surfaces have to be easy to clean and furthermore resistant to water, acid,

alkali, solvents, disinfectants, decontamination agents.

As service ducts and vents usually penetrate the containment barrier, they have to include a separate barrier in form of backflow preventers or HEPA-filters. Backflow prevention for containment laboratories is necessary to prevent recontaminating of liquids and air. Types of backflow solutions are dependent on the medium that is considered.

12

Drainage traps have to be provided to required deep seal depth in consideration of air

pressure differentials. All liquid drains in the facility have to be connected directly

to a liquid waste decontamination system. Drains connected to effluent sterilization

have to be sloped towards the sterilisation system to ensure gravity flow.Supply and

exhaust air ductwork that is outside the containment perimeter (e.g. between contai-

ment perimeter and HEPA filter box or bubble tight backdraft damper) has to be sealed

airtight. The integrity of the filter box containment has to meet the same requirements

as the containment as a whole and has to be tested.

Exhaust and supply HEPA filter housings have to be designed to withstand structural

change at applied pressure (value to be defined).

There has to be provided a system (e.g. fax, computer) for electronic transfer of infor-

mation and date from the laboratory area to outside of the laboratory perimeter.

Decontamination

General:

A personnel body shower with inner and outer changing rooms is required on exit from

the containment laboratory.

Glove-box laboratory:

Complete change of clothing and wet shower including a soaping is required upon exit

from the laboratory. When leaving the laboratory, the laboratory clothing has to be put

in containers, which can be sterilized. The removed clothes remain in the airlock and

are transferred out after sterilization.

Suit laboratory:

A chemical shower of appropriate duration is required for personnel in suits who are

leaving the containment laboratory. A following body shower is not mandatory but

recommended.

13

Effluent:

All effluents from the suit area, decontamination chamber, decontamination shower

or Class III BSC must be decontaminated before final discharge. The preferred decon-

tamination method is heat treatment (autoclaving).

Water from the clean side personnel shower and toilet may be discharged directly to

the sanitary sewer without treatment.

The effluent sterilisation system (e.g. piping, valves, tanks) have to be heat and chemi-

cal resistant consistent with application.

Autoclave:

There must be provided a double-door autoclave to decontaminate waste materials to

be removed from the suit area.

It must be guaranteed by an automatic locking, that the door of the autoclave can only

be opened, when the sterilization cycle has been completed. The barrier autoclave

has to be equipped with visual or audible alarms to prevent both doors from opening

at the same time.

The body of the autoclave is preferably located outside of the containment for ease

of maintenance.

Autoclave condensate drain has to have a closed connection. The condensation water

of the autoclave has to be sterilized prior to be fed to the sewer pipe. A failure of these

sterilization devices has to be prevented by an appropriate design of the valves and by

exhaust valves secured by HEPA filters.

Material can be removed from the containment laboratory only after appropriate de-

contamination. For materials that cannot be autoclaved (e.g. heat sensitive equipment,

samples, films) other proven technologies for waste treatment (e.g. incineration, chemi-

cal, or gas) have to be provided at the containmen barrier.

14

Ventilation System

A dedicated non-recirculating supply and exhaust ventilation system is required.

Supply and exhaust air have to be coupled in a way to prevent an over-pressurization

in case of a failure of the exhaust ventilator.

Exhaust air has to be passed through two stages of HEPA filtration and discharged to

the outside, dispersing the exhaust air away from occupied areas and air intakes.

The filters have to be installed in such a way that their proper functioning can be tested

in place. The supply ducts have to be isolated mechanically behind the filters in order

to allow for a riskless replacement of the filters.

Exhaust HEPA filter housings have to be provided with a method of isolation and de-

contamination. The HEPA filter housings are designed to allow for in situ decontamia-

tion of the filter prior to removal. Alternatively, the filter can be removed in a sealed,

gas-tight primary container (bag-in-bag-out system) for subsequent decontamination

and/or destruction by incineration.

The HEPA filters are located as near as practicable to the source in order to minimize

the length of potentially contaminated ductwork.

All HEPA filters need to be tested and certified annually.

In the containment there has to prevail a negative pressure relative to the pressure of

the immediate environment.

Directional inward airflow has to be provided such that air will always flow towards

areas of higher containment (minimum of 25 Pa differential). An appropriate system

of controls must be used to prevent pressurization of the laboratory: HVAC control

system; supply air system interlocked with the exhaust system; redundant exhaust

fans; appropriate visual pressure monitoring device; alarm.

Emergency power and dedicated power supply lines must be provided.

The laboratory has to be sealed for fumigation. The glove-box laboratory must be at

15

least sealable.

Plumbing vent lines (including effluent sterilization system) have to be provided with

fiters of efficiency equivalent to that of HEPA and provided with a means of isolation

and decontamination.

Suit laboratory:

Redundant supply fans are recommended; redundant exhaust fans are required.

Tertiary Containment

BSL-4 facility design and operational procedures must be documented. The facility

must be tested for verification that the design and operational parameters have been

met prior to operation; facilities must be re-verified annually.

A strict general regulation requires decontamination of any object passing from

the inside of the containment to the outside. This includes materials, waste,

liquids, solids as well as human beings. Animals may never pass through the

containment but must be killed and destroyed by autoclaving, rendering or

incineration. It becomes evident however, that not all objects can be treated

according to the same standards. For material which is not heat resistant but

may not be destroyed, alternative standards have to be applied. Furthermore,

personnel can only be decontaminated to a certain degree, therefore when

working with organisms of risk group 4 a BSC Class III or a full body suit is

required. However, it must be recognized that the term “sterile” is not always

adequate for all objects leaving the containment. The term “decontamination”

is used instead. This has to be communicated very carefully to lay persons

when it becomes an issue. The term “sterile” historically comes from the

pharmacopoeia which defines sterility of pharmaceuticals. However, it does

not seem to be adequate to apply the same rules to sewage water for exam-

ple. Therefore, a more adequate and pragmatic solution must be found in the

near future to overcome this uncertainty in risk.

16

The containment laboratories have to be located away from external building envelope

walls; furthermore they must be separated from other areas in the same building or

be situated in a separate building.

Access to the facility has to be limited by means of secure, locked doors to persons,

whose presence in the facility is required for program or support purposes; accessi-

bility is managed by the laboratory director, biohazard control officer, or other person

responsible for the physical security of the facility.

A biohazard sign on the door must identify the agent, list the name of the responsible

person(s), and indicate any special requirements for entering the area (e.g. need for

immunizations or respirators).

Personnel entering the laboratory must remove street clothing and jewelry, and change

into dedicated laboratory clothing and shoes.

Conclusions

The analysis demonstrates that the complex and wide variety of requirements can easily

be reduced to a set of general rules covering all the national and international specific

rules and therefore representing the state of the art of containment construction.

17

Bibliography

[1] Holeman, Penny/Gueltekin, Halil: An Overview: Biological Safety from a Global

Perspective. In: Richmond, Jonathan (ed.): Anthology of Biosafety. Vol. III,

Application of Principles. ABSA 2000. p. 83.

[2] US Electronic Code of Federal Regulations (e-CFR) current of October 26,

2005 (download October 28, 2005): 32 CFR 627.16; 32 CFR 627.46.

[3] EU Council Directive 98/81/EC, Annex IV: Containment and Other Protective

Measures.

[4] Swiss Ordinance on the Contained Use of Organisms (Verordnung über den

Umgang mit Organismen in geschlossenen Systemen [Einschliessungsverord-

nung, ESV], SR 814.912).

[5] German GMO Guideline (Verordnung über die Sicherheitsstufen und Sicher-

heitsmassnahmen bei gentechnischen Arbeiten in gentechnischen Anlagen

[Gentechnik-Sicherheitsverordnung - GenTSV]).

[6] WHO Laboratory Biosafety Manual, Third Edition. Chapter 5: The Maximum

Containment Laboratory - Biosafety Level 4. Geneva 2004.

[7] Health Canada: Laboratory Biosafety Guidelines, Third Edition, 2004.

[8] CDC/NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL), 4th

Edition. Washington 1999.

Appendix x

Appendix x

Appendix x

Appendix x