Sacramento

January 28, 1977

Testimony/California State Legislature
Maxine Singer

As a result of the International Conference on Recombinant DNA at
Asilomar, California in February 1975, activities were initiated in every
country in the world where scientific capability permits the experiments.
These activities were, and are, directed toward providing assurance that
potentially hazardous organisms, constructed by the new techniques, will not
be inadvertently released. It is important to recognize that these activities
are proceeding in the absence of any demonstration that hazardous organisms
can indeed result from the experiments.

On June 23, 1976, Donald S. Fredrickson, Director, National Institutes
of Health, announced publication of guidelines designed to eliminate or
minimize any potentially hazardous consequences of recombinant DNA research.
The guidelines were subsequent!y published in the Federal Register (Part ||
for 7 July 1976).

The promulgated guidelines were the result of a year and a half of
intensive work by the NIH Recombinant DNA Molecule Program Advisory Committee
as well as consideration by the Director of a variety of views expressed by
individuals and private organizations either in writing or at a public hearing
in February 1976. A summary of the history of the development of the guile-
lines as well as the various views expressed by many commentators is available
in the Director's Decision Statement, which accompanied publication of the
guidelines.

A Draft Environmental Impact Statement was prepared by the NIH, after
determining that publication of the Guidelines was a "major federal action"

as defined by the National Environmental Policy Act of 1969. The Draft
Statement was published in the Federal Register of September 9 1976, and
circulated widely. Comments on the Draft Statement were received and exten-
sively reviewed by the scientific and administrative statts of the NIH. And
the final Environmental Impact Statement is now being prepared.

For the purposes of the guidelines recombinant DNA experiments are
defined as those involving molecules that consist of different segments of
DNA which have been joined together in cell-free systems, and which have the
capacity to infect and reproduce in some host cell, either autonomously or
as an integrated part of the host's genome.

In the experiments discussed in the guidelines the host cells are
generally single living cells, either microorganisms such as bacteria, or
animal or plant cells grown as single cells in tissue culture.

The guidelines start with a statement of general principles and these
are consistent with the conclusions published in the report of the Conference
at Asilomar. The first principle is that there are certain experiments which,
in the light of currently available information, may be judged to present
potential hazards of so serious a nature, should they prove to be hazardous
that they should not be attempted at this time. Second, a large group of
feasible experiments appear to pose lesser or no potential hazard, and can
therefore be performed provided that the information to be obtained, or the
practical benefits anticipated, cannot be obtained by conventional methods,
and provided that appropriate safeguards for containment of potentially
hazardous organisms are incorporated into the design and execution of the
experiment. Third, that the more serious the nature of any possible

hazardous event, the more stringent should be the safeguards against escape
of the potentially hazardous agents. The safeguards should be at least as
stringent as those generally used to handle the most hazardous parent of the
recombinant. Since the estimation of potential hazards is conjectural and
speculative, the levels of containment required for potentially hazardous
organisms should be set high initially, and modified only when there is
substantial relevant information to advise such modifications. And finally
that the guidelines are to be reviewed at least annually in order to account
for new information.

Containment Methods: Three approaches to the problem of containing
potentially hazardous organisms form the basis of the recommended safeguards.
Each of the three set up barriers to the dissemination of potentially hazardous
organisms from the laboratory situation, and barriers between the laboratory
worker and the organisms. Two of these approaches involve the limitation of
the actual physical escape of the organisms, and are referred to as physical
containment. The first such approach is the set of standard microbiological
practices, that have been developed over a period of many years, and are
widely used for handling pathogenic organisms both in research and clinical
laboratories. in the hands of well trained personnel, these procedures have
proven to be effective in safeguarding both the worker and the environment
from the spread of many pathogenic agents. The second approach to physical
containment involves the use of special kinds of equipment and facilities
- to limit spread of aerosols, - for decontamination and containment of
laboratory air and wastes, and for limitating access to laboratories. As
with the standard microbiological techniques the type of equipment and

facilities are not new, but were developed and have been used for containment
of known pathogenic organisms. There is documented experience on which to
judge the efficacy of the various physical barriers in preventing the escape
of organisms.

The guidelines go into some detail concerning the practices and
facilities required for physical containment: four levels of physical con-
tainment are specified. They are termed P1, P2, P3 and P4 in the order of
increasing levels of containment. P1, the lowest level, consists of the use
of the standard microbiological practices mentioned before. The P2 and the
next higher level P3, each require special procedures and facilities (including
vertical laminar flow biological safety cabinets and laboratories maintained
at lower air pressure than the surrounding building) all designed to limit to
increasing extents any possible accidental escape of potentially hazardous
organisms. Finally, P4, the maximum level of containment requires sophisticated
and isolated facilities designed for maximum containment. Each of the levels,
P2, through P4, assumes that the techniques demanded by P1, the standard
microbiological practices, will be followed. Furthermore, for each level,
relevant training of personnel is mandatory. The training is to include the
nature of the potential hazards, the technical manipulations, and instruction
in the biology of the organisms and systems. Specific emergency plans, to
be used in case of accident, are required and serological monitoring, where
appropriate, is to be provided.

The third approach to the problem of containing potentially hazardous
organisms within the laboratory is the use of biological barriers. Biological
containment is defined as the use of host cells and vectors with limited

ability to survive outside of very special and fastidious laboratory conditions.
These conditions are unlikely to be encountered by escaped organisms in
natural environments. Biological containment is an integral part of the
experimental design, since the host and vector will need to be chosen,

in any given experiment, with a view both to the purpose of the experiment
and to containment. The guidelines stress that physical and biologic
containment procedures are complementary to one another each one serving
to control any possible failure in the other. The use of both, in a given
exper Imesfht fords much higher levels of containment than either one alone.
Therefore, the guidelines always recommend both a particular level of
physical containment, and a level of biological containment for any given
experiment.

The guidelines also recommend that publications describing work on
recombinant DNA include a description of the containment procedures used.
The guidelines then classify all likely experiments, rank them according
to potential hazard and assign the required containment fevels. The first
class of experiments described in the guidelines are those which are
prohibited. These are experiments which are judged to be of potentially
very serious hazard, should they in fact be hazardous. The prohibited
experiments include the following.

(1) Any experiment in which a portion of the recombinant DNA derives
from either a highly pathogenic organism ~ an organism that causes serious
disease in man or agriculturally important plants or animals - and classified
as such by the Center for Disease Contro!, of the United States Public Health
Service, or derives from cancer causing viruses classified by the National

Cancer Institute as moderate risk. Perhaps | should stress here that these
viruses cause cancer in animals. There is no virus known to cause cancer
in humans.

(2) Deliberate formation of recombinants containing the genes for
toxins of very high toxicity. Examples of this class are botulinus toxin
or diphtheria toxin, and venoms from insects and snakes.

(3) Deliberate creation from plant pathogens of recombinant DNAs that
are likely to increase either the virulence of the pathogenic material or
the range of species susceptible to the disease.

(4) Certain of the possible beneficial applications of DNA recombinant
research involve the creation of organisms with the ability to carry out
useful environmental functions. Release of such organisms into the environ-
ment may at some point be required to test their efficacy, and certainly
to make use of them. Nevertheless, the guidelines state that deliberate
release of any organism containing a recombinant DNA molecule is not to be
undertaken at present.

(5) Transfer of a drug resistance trait to microorganisms that are not
known to acquire the trait naturally if such acquisition could compromise
the use of the drug to control disease agents in human or veterinary medicine
or agriculture.

(6) Finally experiments must be limited in scale to quantities of
fluid less than 10 liters with recombinant DNAs known to make harmful products.
The guidelines state that the Advisory Committee may make exceptions to this
rule for particular experiments deemed to be of direct societal benefit if
appropriate equipment is used.

The classification of permissible experiments is first made on the basis
of the host-vector system that will be used. Recognizing the relation

between the host-vector system required by the experiment and the design of
suitable biological containment, experiments using the same host-vector

system are grouped together. Then the guidelines classify permissible
experiments according to the best available estimates of potential risk.

In the absence of much needed data, these estimates involve informed judgment
in many instances. For example, not all recombinant DNA experiments yield
novel combinations of DNA....recombination between the DNAs of organisms

known to exchange genetic information in nature do not add uniquely man made
cells to the biosphere. In these cases, the guidelines follow the principle
that the experiments are to be carried out under conditions generally used

to handle the most hazardous parent of the recombinant. When DNAs from species
not known to exchange genetic material in nature are recombined, more stringent
and strictly defined containment is required. Moreover, in many such experi-
ments it is mandatory to use biologically contained agents that have been
certified by NIH as unlikely eitner to propagate outside of rigorously defined
laboratory environments or to transfer the recombined DNA to other cells.

These agents include certain derivatives of a bacterial species called E. coli

 

Strain K12. Strain K12 has been studied extensively for 30 years and can be
readily manipulated for recombinant DNA experiments. This same extensive
experience and ease of manipulation permits modification of E. coli K12 and
its vectors by classical genetic techniques, for the purpose of establishing
Diological containment.

The nature and manner of achieving biological containment with this

system is described in the guidelines. E. coli K12 appears to be harmless

 
itself, it does not usually either establish itself or multiply signifi-
cant!y in the normal bowel. These facts suggest that accidental injestion
of a small number of bacteria by a laboratory worker would not result in
extensive spread of the bacterium outside the laboratory. The normal situation
may be altered when people are either taking antibiotics, or have certain
abnormal digestive conditions and it is required that such individuals refrain
from work for the duration of the abnormal situation. However, while E. coli
K12 does not establish itself as a growing strain in normal bowels, it does
remain alive during its passage through the tract. Therefore transfer of
plasmid or bacteriophage vectors containing foreigh DNA from the original
E. coli K12 host to bacteria resident in the intestines or bacteria encountered
after excretion must be considered. For any given E. coli K12 host-plasmid
combination used in a recombinant DNA experiment it will be necessary to assess
the possibilities for transfer of the recombinant DNA in order to evaluate the
degree of biological containment. While we are missing some relevant infor-
mation, the available data suggest that the probability of transfer can be quite
low, depending on the particular system.

The use of this bacteria has caused wide concern...and certain facts need
to be emphasized. Only one strain of E. coli, called K12, is permitted by
the guidelines. Strain K12 is one of a large group of bacterial strains, al!
of which are called E. coli because they share certain properties in common.
But they do not all have identical properties. Some E. coli live normally in
the intestines of healthy people and animals: others are pathogens, that is,
disease producers. K12, which is rarely found in nature, and does not

normally colonize human or animal intestines, is a greatly enfeebled strain
whose principle successful ecological niche is in the laboratories of
molecular biologists and geneticists. It is not pathogenic. If it were,

you and | would not be here worrying about this research, since all the
molecular biologists would long since have disappeared. Pathogenicity is

a complex phenomenon....dependent on several properties of the pathogen as
well as on the properties of the species being infected. It is very unlikely
that alterations in K12 brought about by insertion of recombined DNA wil!
make it into a pathogen....but it is not impossible. I+ is this remote
possibility with which we are all concerned. We are attempting to protect
against an unlikely, uncertain, yet unacceptable event. Thirty years of
studey of the genetic chemistry of E. coli strain K12 provides confidence
that the capacity of these bacteria to escape and spread in the environment
can be reduced to immeasurable levels. Thus, should pathogenic organisms
arise, it is not likely they would survive to cause disease....nor is it
likely that bacteria containing recombined DNA would survive to evolve in
unique and fearsome ways. Nevertheless, because of K12s relation to common
strains of E. coli, reservations about its use persist. It its important to
investigate alternative organisms...and the Guidel ines encourage this but it
is not at all certain that useful and safer bacteria exist. Predictions about
the existence of rare and fastidious organisms, unable to exchange DNA with
bacteria inhabiting man or other living things, are highly speculative.
Considering then the properties of E. coli K-12, as well as those of the
certain known plasmid and bacteriophage vector, the guidelines conclude that,
using such host-vector systems, recombinant DNAs are unlikely to be spread

by the ingestion or dissemination of the few hundred or thousand bacteria,
such as might be involved in laboratory accidents, given standard micro-~
biological practice. Therefore, these existing systems, are judged to offer
a moderate level of biological containment and are defined as EK1, the lowest
level of biological containment for experiments with E. coli systems.

As with physical containment levels, increasing numbers specify
increasing levels of biological containment for E. coli systems. The next
level is called EK2. EK2 host-vector combinations must be demonstrated to
provide a high level of biological containment by suitable laboratory tests.
They are obtained by genetic modification of either existing E. coli K12
host cells or the relevant vectors or both. More specifically, the guidelines
state that in order to qualify as EK2 the modified system composed of deri-
vatives of E. coli K12 combined with a particular vector should not permit
survival of the vector in other than specially designed laboratory environ-
ments at a frequency greater than 10/5100 million). Various examples of the
types of necessary modifications are suggested in the guidelines.

One additional level of contained E. coli host-vector systems is defined
in the guidelines and is called EK3. EK3 systems are EK2 systems for which
the specified containment properties have been demonstrated not only by
microbiological and genetic anaylsis but by appropriate tests in animals
including humans or primates and other relevant environments.

EK2 and EK3 host vector systems must be recommended for approval! by
the Recombinant DNA Program Advisory Committee and then certified by the
Director, NIH before use. Detailed data on the relevant properties of the
system must be submitted for consideration by the Committee. Thus far,

several EK2 systems have been approved and certified. No EK3 systems have
been submitted for certification as yet.

Having ranked experiments according to potential hazard, and defined
the several levels of physical containment and biological containment the
guidelines then assign to each type of experiment both a required physical
containment level, that is a P level, anda required biological containment
level, that is an EK level and the particular combination of the two reflects
the severity of the estimated potential hazard. The Guidelines are organized,
for the E. coli systems, according to the source and nature of the foreign
DNA. In general it is assumed that potential hazard increases with increasing
relatedness of the foreign DNA to man. EXperiments in which the foreign DNA
is highly purified, and does not contain harmful genes are assigned lower
containment requirements than those in which the foreigh DNA is an unpurified
mixture of fragments - the so-called shotgun experiments. Experiments
involving such mixtures of DNA fragments are assumed to be of relatively
higher potential hazard because of the greater likelihood of dangerous and
unknown genes being introduced into a recipient cel! compared to experiments
with a single, highly purified fragment.

In addition to experiments using E. coli K12, the Guidelines specify
containment conditions for certain other host-vector systems. Many recom-
binant DNA experiments will involve the use of systems in which the host
cells are derived from animals or plants but are grown as single cells in
the laboratory. Given the current state of technology, DNAs from animal
viruses are most likely to be used as vectors in the near future. The cells
themselves are fragile and fastidious and there is little or no chance that

a living cell could escape from a laboratory in the way that an E. coli
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cell might. Therefore containment considerations focus on the viruses since
they might escape a laboratory in a viable form. There are two animal viruses
whose DNAs are, now, technically useful as vectors; polyoma and simian virus
40 (SV40). In their respective normal hosts, mouse for polyoma, rhesus
monkeys for SV40, neither virus causes a known disease. Polyoma does not
infect human cells grown as single cells in the lab and also does not appear
to infect humans, since humans exposed to polyoma do not produce antibodies.
SV40 does infect both human cells grown as single cells in the laboratory,
and whole human beings, as evidenced by the active production of antibodies
and the reports of isolation of SV40 from humans. This virus contaminated
the early Salk polio vaccines and millions of people were inadvertently
inoculated with it in the middle 1950s. To date, there is no indication that
the recipients of the vaccine suffered any related difficulty. Both polyoma
and SV40 cause tumor formation in newborn small laboratory mammals. They are
classified as low risk oncogenic viruses by the National Cancer Institute.
Because SV40 infects human beings, and also because SV40 and related viruses
nave been isolated In connection with several human disease states, the
proposed guidelines assume that polyoma inherently affords a higher level of
biological containment: therefore more stringent physical containment is
required for SV40 than for polyoma.

The guidelines require that the viral DNA used for recombination with
a foreign DNA must itself be defective - in a manner analogous to the EK2
systems. Physical containment conditions are specified, and these depend,
as before, on the nature of the "foreign" DNA fragment to be spliced onto the
viral DNA. Experiments with polyoma and SV40 are restricted to P3 and P4

levels only.
13

The Guidelines also contain recommendations for experiments in which
plant cells will serve as hosts for recombinant DNA. The cells might be
single plant cells grown under laboratory conditions, or seedlings, plant
parts, or small whole plants. This is in fact the only instance where the
guidelines address the question of recombinant DNA experiments with whole
organisms. Directions are given for modification of the specifications for
P1, P2, and P3 physical containment in order to provide conditions appropriate
for work with plants.

Implementation of the Guidelines. The guidelines contain a large section
defining the roles and responsibilities of individuals and institutions in
assuring compliance with required containment levels. The procedures, as
described, are primarily directed at grantees of the National Institutes of
Health. Similar procedures are in force for work carried within the NIH
laboratories themselves, and for work carried out under contract arrangements
with the NIH.

The principle investigator is required to assess any potential
biohazards, to institute appropriate safeguards and procedures, to minimize
effects of possible accidents by planning, to train and inform all personnel,
to report any serious or extended illness of a worker or any accidents, and
all of these must be carried out on a continuing basis. Thus, the primary
responsibility for conducting experiments according to the guidelines is in
his hands. Further, in applying for grants to carry out experiments with
recombinant DNA, the investigator must include an estimate of the potential
biohazards as well as a statement as to the containment procedures that wil|
be used. The application must include certification as to the existence and

availability of appropriate facilities, procedures, and training. The
14

guidelines indicate that institutions in which recombinant DNA exper iments

are carried out must establish biohazard committees which can serve to examine
equipment and facilities and certify their compliance with the requirements.
Such committees will also serve as a source of advice and reference on
physical containment facilities, on properties of biological containment,

and on training of personnel.

According to the proposed guidelines review of the certification and
of the investigators judgment concerning the extent of potential hazard and
the required containment would be by NIH study sections, during the normal
scientific review of the application. After approval, but before work is
initiated, the investigator must formalize his commitment to abide by the
Guidelines by submitting to the NIH a memorandum of understanding. Both
the investigator and a responsible official of his institution must sign this
document.

Failure, on the part of an investigator, to comply with the provisions
of the guidelines, may result in cancellation of his research grant by the
NIH. The NIH has no statutory regulatory authority and is thus limited in
the sanctions it can use.

Application of the guidelines to work not supported by the National
Institutes of Health. Several agencies of the U.S. government other than the
National Institutes of Health provide support for biological and medical
research and are currently, or may in the future, sponsor recombinant DNA
experiments. Ail of these agencies have now adopted the NIH Guidelines and
will apply them to all research under their sponsorship. These include the
National Science Foundation, the Energy Research and Development Adminis-

tration, the Department of Defense, the Department of Agriculture, and
15

the National Aeronautics, Space Administration. At present there are no
mechanisms except voluntary ones for extending the provisions of the
guidelines to work supported by private funds - either for research or
commercial purposes or to work supported by state and local funds. Both
the executive and legislative branches of the federal government as well
as concerned private individuals and organizations have recognized the
urgent need to establish such a mechanism.

A federal Interagency committee, chaired by the Director of NIH, and
formed at the request of the President is at work. Both research and
regulatory agencies are involved. The Committee's charge was to determine
whether existing powers within the Federal regulatory agencies, are sufficient
to extend control to the non-federal sector....to formulate recommendations
as to how this may best be done, and to recommend legislation should that
be deemed necessary. The Committee has not yet completed its work, but |
can report to you on its progress to date. A careful examination of existing
regulatory authorities within the EPA, the OSHA, the Center for Disease Control,
and the FDA has been made. I+ is probable that none of the existing authorities
can properly cover the particular requirements of the recombinant DNA situa-
tion. The NIH itself has no regulatory authority. Thus the Interagency
Committee may conclude that new legislation will be required. In that instance
an appropriate legislative framework will need to be devised. The various
concerned Federal agencies may be expected to consult with their particular
constituencies for advice on ways to proceed - the Department of Commerce with
interested industry - the Department of Labor with Unions, the Department of

- Agriculture with agricultural interests and the NIH with scientific community.
16

A high level of interest in the Congress is stimulating the Interagency
Committee to prompt resolution of this problem: we may therefore expect
action to extend the applicability of the guidelines - or some modification

of them - to all privately or locally sponsored work with recombinant DNA

in this country.