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  Summary Statement of the Asilomar

Conference on Recombinant DNA Molecules

I.

Int r oduc t ion and Gene r a1 C onc lu s ions

This meeting was organized to review scientific progress in

the research on recombinant DNA molecules and to discuss appropriate

ways to deal with the potential biohazards of this work.

heard about the impressive achievements already made in this field

and seen glimpses of the remarkable potential of these methods for

furthering our understanding of the fundamental processes in

eukaryote cells.

to revolutionize the practice of molecular biology.

as yet been no practical application of the new techniques,  there

is every reason to believe that they will have significant impact in

the future.

We have

The use of recombinant DNA methodology promises

While there has

Of particular concern to the participants of the meeting was

the issue of whether the pause in research in this area,  called for

in the letter published in July, 1974, by the Committee on Recombinant

DNA Molecules of the National Academy of Sciences, U. S. A.,  should

end;  and, if so, whether there are ways in which the scientific work

can be undertaken with minimal risks to workers in laboratories,  ta

the public at large and to the animal and plant species sharing our

ecosystems. It was emphasized that, in the longer term, even more

difficult problems may arise in the large scale application of this

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work in industry, medicine and agriculture. Even in'the present,

more limited conduct of research in this field, the evaluation of

potential biohazards has proved to be extremely difficult.

The new

techniques permitting combination of genetic information from very

different organisms place us in an area of biology with many unknowns.

It is this ignorance that has compelled us to conclude that it would

be wise to exercise the utmost caution.  Nevertheless, it was agreed
that@ of the work on construction of recombinant DNA molecules
e proceed but with appropriate safeguards, principally, biologic

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and physical barriers adequate to contain the newly created organisms.

Further, the participants of the Conference concluded that there are

certain experiments in which the potential risks are of such a serious

nature that they ought not to be done with presently available con-

tainment facilities.  Future research and experience may show that

many of the potential biohazards are less serious and less probable

than we now suspect.

protection should be greater at the beginning and modified as assess-

ments of the risks change. Moreover, an anticipated change in

experimental design causing increased risks,

However, it was agreed that standards of

however small,

should be carefully evaluated,

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11.

Principles Guiding the Recommendations and Conclusions

    Though our assessments of the risks involved with each

of the various lines of research on recombinant DNA molecules

may differ, few, if any, believe that this methodology is free from

any risk.

risks are: 1) that containment be made a significant consideration

of the experimental design and;  2)  that the effectiveness of the

containment should match as closely as possible the estimated risk.

Consequently, whatever scale of risks is agreed upon, be it

a three, four or multi-category classification, there should be a

commensurate scale of containment.

will be difficult and empirical at first but this will improve

as we acquire additional knowledge;

match the potential risk with an appropriate level of containment.

Reasonable principles for dealing with these potential

Estimating the risks

at each state we shall have to

Experiments requiring large scale operations would seem to be

riskier than equivalent experiments done on a small scale and, there-

fore, require more stringent containment procedures.

v

the use of cloning vehicles or vectors (plasmids, phages) and bacterial

hosts with a restricted capacity to multiply outside of the laboratory

would increase the safety of a particular experiment.
which potential biohazards and different levels of containment are

matched may vary from time to time particularly as the containment

technology is improved. Quite likely the means for assessing and

balancing risks with appropriate levels of containment will need to

The ways in

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be reexamined from time to time.

and informal channels of information within and between the nations

of the world, the way in which potential biohazards and levels of

containment are matched would be consistent.

Hopefully, through both formal

Containment of potentially biohazardous agents can be achieved

in several ways.  The most important, because it contributes most

significantly to limiting the spread of the recombinant DNAs,  is the

use of biological barriers.  These barriers are of two types:
Fastidious bacterial hosts unable to survive in natural environments,

and 2) non-transmissible and equally fastidious vectors (plasmids,
bacteriophages or other viruses) able to grow only in specified hosts.

Physical containment, exemplified by the use of suitable hoods,  or,

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where applicable, limited access or negative pressure laboratories,

provides an additional factor of safety. Additionally, adherence to

good microbiological practices which to a large measure can limit

the escape of organisms from the experimental situation, contributes

measurably to the safety of the operation.

C onsequently,education and

training of all personnel inv.olved in the experiments is essential to.

the effectiveness of all cantaixment measures,

In practice the

different means of containment will complement one another and

substantiated improvements in the ability to restrict the growth of

bacterial hosts and vectors could permit modifications of the com-

al

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plementary containment requirements .

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Stringent physic a1 containment and rig or ous labor ator y

procedures can reduce but not eliminate the probability of spreading

potentially hazardous agents e

upon "disarmed" bacterial hosts and vectors for additional safety

Therefor e, inve stig ator s relying

must test rigorously the effectiveness of these agents before accept-

ing their validity as biological barriers.

111.

Specific Recommendations for Matching Types of Containment with

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Types of Experiments

No classification of experiments as to risk and no set of

containment procedures can anticipate all situations.

Given our

present uncertainties about the hazards, the parameters proposed

here are broadly conceived and meant to provide provisional guide-

lines for investigators and agencies concerned with research on

recombinant DNAs. But eachinvestigator bears a responsibilityfor

determining whether, intheir particular case, special circumstances

warrant greater care thanis suggestedhere.

A. Types of C ontainment

  1. Minimal Risk: This type of containment is applicable

experiments in which the biohazards may be reasonably assessed

to

and are expected to be minimal.

Such containment can be achieved

by following the operating procedures recommended for clinical

microbiological laboratories.

Essential features of such facilities

are a ban on drinking, eating or smoking in the laboratory, wearing

laboratory coats in the work area, the use of cotton-plugged pipettes

or preferably mechanical pipetting devices and prompt disinfection

of contaminated materials.

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- 2. Low Risk: This level of containment is appropriate for

, experiments which generate novel biotypes but where the available
information makes it unlikely that the recombinant DNA can alter
appreciably the host's ecological behavior, increase significantly
its pathogenicity, or prevent effective treatment of any resulting

infections.
minimal procedures mentioned above) are a ban on mouth pipetting,
access limited to laboratory personnel, and the use of biological
safety cabinets for procedures likely to produce aerosols (e. g.,

The key features of this containment (in addition to the

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experiments in which there is a probability of generating an agent

# significant$-3 potential for pathogenicity or ecological

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The principle features of this level of containment, in
those of the two preceding classes, are that transfer
should be carried out in biological safety cabinets (e. g.,

laminar flow hoods), gloves should be worn during the handling of

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infectious materials, vacuum lines and drains must be protected by

filters and negative pressure should be maintained in the limited

access laboratories. Moreover, such experiments must be done

only with vectors and hosts that have an appreciably reduced capacity
to multiply outside of the laboratory.

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4. High Risk: This level of containment is for experiments

in which the effects on the ecological potential or pathogenicity of the

modified organism could be severe and thereby pose a serious bio-

hazard to the investigator or the public at large.

of this facility, which was designed to contain highly infectious

microbiological agents, are its isolation from other areas by air

locks, a negative pressure environment, a requirement for clothing

changes and showers for entering personnel and laboratories fitted

with treatment systems to inactivate or remove biological agents that

may be contaminants in exhaust air, liquid and solid wastes.  All

persons occupying these areas should wear protective laboratory

clothing and shower at each exit from the containment facility.  The

handling of agents should be confined to biological safety cabinets in

which the exhaust air is incinerated or passed through Hepa filters.

High risk containment includes, beside the physical and procedural

features described above, the use of rigorously tested vectors and

hosts whose growth can be confined to the laboratory.

The main features

B. Types of Experiments

Accurate estimates of the risks associated with different

types of experiments are difficult to obtain because of our ignorance

of the probability that the anticipated dangers will manifest them-

selves. Nevertheless, experiments involving the construction and

propagation of recombinant DNA molecules using DNAs from 1)

prokaryotes, bacteriophages and other episomes, 2) animal viruses,

and 3) eukaryotes have been characterized as minimal, low,

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moderate and high risks to guide investigators in their choice of the

appropriate containment.
interim assignments which will need to be revised upward or down-

ward by future experience.

These designations should be viewed as

The recombinant DNA molecules themselves,  as distinct

from cells carrying them, may be infectious to bacteria or higher

organisms. DNA preparations from these experiments, particularly

in large quantities, should be chemically inactivated before disposal.

   1. Prokaryotes, bacteriophage,s and other episomes: Where
the construction of recombinant DNA molecules and their propagation

prokaryotic agents that can normally exchange genetic
  the experiments can be performed in minimal risk

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containment facilities. Low risk containment facilities should be

used where the elements comprising the recombinant DNA molecules
are derived from agents that ordinarily do not exchange genetic

information.

pathogenic organisms or the creation of organisms with increased

pathogenicity for man or the environment, a moderate or high risk

containment should be used depending upon the severity of the

potential hazard.

resistance of the recipient species or otherwise compromising the
controllability of known pathogens should be undertaken only under
moderate or high risk containment depending upon the virulence of

the organism involved.

-

If the experiments involve genetic determinants from

Experiments extending the range of antibiotic

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2. Animal Viruses: Experiments involving linkage of

viral genomes to prokaryotic vectors and their propagation in

prokaryotic cells should be performed only with moderate risk con-

tainment c apability .

Rigorously purified and char act erized segments

of non-oncogenic viral genomes or of the demonstrably non-transforming

regions of oncogenic viral DNAs can be attached to existing vectors

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sing moderate containment facilities.

Experiments designed to introduce or propagate DNA
iral or other low risk agents i3 animal cells should use

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k animal virus DNAs as vectors and manipulations should

be confined to moderate risk containment facilities.

3. Eukaryotes: The risks associated with joining random

fragments of eukaryote DNA to prokaryotic DNA vectors and the

propagation of these recombinant DNAs in prokaryotic hosts are the

most difficult to as s e s s .

A priori, the DNA from warm-blooded vertebrates is

more likely to contain cryptic viral genomes potentially pathogenic

for man than is the DNA from other eukaryotes.

Consequently

attempts to clone segments of DNA from such animal genomes shoulci

be performed in a moderate risk containment facilit

vector-host system available4 Until cloned segments of warm-

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characterized, they should

continue to be maintained in the safest vector-host system in

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Unless the organism makes a product known to be

dangerous (e. g., toxin, virus), recombinant DNAs from cold-blooded

vertebrates and all other lower eukaryotes can be constructed and
propagated with the safest vector-host system available in low risk

containment facilities.

Purified DNA from any

source that performs known

functions and can be judged to be non-toxic, may be cloned with
currently available vectors in low risk containment facilities.
(Toxic here includes potentially oncogenic products or substances
that might perturb normal metabolism if produced in an animal or
plant by a resident microorganism. )

4. Experiments To Be Deferred: There are feasible experi-

ments which present dangers serious enough that their performance

e this timgshould not be undertaken with the presently available pb3ab rl.IC

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con?ai-ent ' & apability, 5.

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These include the cloning of recombinant DNAs derived from highly
pathogenic organisms, DNA containing toxin genes and very large
scale experiments using recombinant DNAs that are able to make
products potentially harmful to man, animals or plants.

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IV. Implementation

    In many countries steps are already being taken

bodies to formulate codes of practice for the conduct of

by national

experiments

with known or potential biohazard. Until these are established, we

urge individual scientists to use the proposals in this document as

a guide. In addition, there are some recommendations which could

be immediately and directly implemented by the scientific community.

A.

Development of Safer Vectors and Hosts

    One of the most important and interesting accomplishments

of the meeting was the realization that special bacteria and vectors

can be designed and constructed and that the use of these could

radically enhance the safety of experiments by many orders of magni-

tude.

certain that in the near future there will become available variants

of X bacteriophage, non-transmissible plasmids and special strains

Appendix G contains a summary of the suggestions;

it is

of E. coli all of which could in principle, reduce biohazards by

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very large factors, as well as bringing about technical improvements

in the methods themselves. Other bacteria, particularly mutably

modified strains of B. subtilis, may also be specially useful for

particular purposes.

vector may be found for simple eukaryotic cells such as yeast.

There should be a continuous development in this area and the parti-

cipants at the meeting agreed that all such strains will be made

freely available.

There is also the possibility that a suitable

B.

Lab or ator y Pr oc edur e s

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It is the clear responsibility of the principal investigator to

inform the staff of the laboratory of the hazards of such experiments,

before they are initiated.

Free and open discussion is necessary so

that each individual fully understands the nature of the experiment

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All workers must be properly trained in the containment procedures

that are designed to control the hazard, including the emergency

action in the event of a hazard.

priate health surveillance of all personnel, including serological

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It is also recomrnended that appro-

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monitoring, be conducted periodically.

C, Education and Reassessment

Research in this area will develop very quickly and the methods

will be applied to many different biological problems.

It is impossible

at any given time to foresee the entire range of all potential experi-

ments and make judgments on them.

It is therefore essential to

undertake a continuing reassessment of the problems in the light of

new scientific knowledge.

This could be achieved by a series of

annual workshops and meetings, some of which should be at the

international level,

There should also be courses to train individuals

in the relevant methods since it is likely that the work will be taken

up by laboratories which may not have had extensive experience in

this area.

Consideration should also be given to the establishment of

ertinent to the effectiveness of biological containment,

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V. New Knowledge

J

This document represents our first assessment of the potential

biohazards in the light of current knowledge.
about the survival of laboratory strains of bacteria and bacteriophages

in different ecological niches in the outside world.

known about the possibility of recombinant DNA molecules to enhance

or depress the selective value of their vectors in Nature.

questions are fundamental to the testing of any new organisms that

may be constructed.  Research in this area will have to be undertaken

and should be given high priority.  The difficulty is that, in general,

molecular biologists who may construct DNA recombinant molecules
do not undertake these experiments and it may be necessary to
facilitate collaborative research between them and groups skilled in
the study of bacterial infection or ecological microbiology.
work should also result in methods to monitor any possible escape

or dissemination of cloning vehicles.

Very little is known

Even less is

These

Such

Nothing is known about the potential infectivity in higher

organisms of phages or bacteria containing segments of eukaryotic
DNA and very little about the infectivity of the DNA molecules thern-

selves.  Genetic transformation of bacteria does occur in animals

suggesting that DNA molecules can retain their biological potency

in this environment.
%!the answers to which are essential for our assessment of the

biohazards of experiments with recombinant molecules.  It will be

necessary to ensure that this work will be planned and carried out;

There are a large number of questions in this

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and it will be particularly important to have this information before

largescale applications of the use of recombinant molecules is

attempted .