1

Sacramento
January 28, 1977
Testimony/California State Legis
Maxine Singer

ature

As a result of the lnternat onal 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

can indeed result from the exper

   On June 23, 1976, Donald S.

of Health, announced publication

minimize any potentially hazard0

any demonstration that hazardous organisms

ments


Fredr

of gu


s con

ckson, Director, National Institutes

delines designed to eliminate or

equences of recombinant DNA research.

The guidelines were subsequently published in the Federal Register (Part II

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.

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 summary of the history of the development of the guile-

   A Draft Environmental  Impact Statement was prepared by the NIH,  after

determining that publication of the Guidelines was a %ajar federal  actiont1

as defined by the National Environmental Policy Act of 1969. The Draft

2

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 staffs 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.

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.

hazardous event, the more stringent should be the safeguards against escape

The first principle  is that there are certain experiments which,

Third, that the more serious the nature of any possible

3

of the potent

str i ngent as

recombinant.

        hose generally used to handle

        Since the estimation of poten

speculative, the levels of containment requ

organisms should be set high initially, and

ally hazardous agents. The safeguards should be at least as

the most hazardous parent of the

ial hazards is conjectural and

red for potentially hazardous

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 informat ion.

  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

organ isms

worker and


the actual


containmen

practices,

rom the laboratory situation, and barriers between the laboratory

the organisms. Two of these approaches involve the limitation of

physical escape of the organisms, and are referred to as physical

. The first such approach is the set of standard microbiological

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.

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

The second approach to physical

4

of known pathogenic organisms.

judge the efficacy of the various physical barriers in preventing the escape

of organ isms.

There is documented experience on which to

  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 Pl, 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.  Spec

be used in case of accident, are required and sero

appropriate, is to be provided.

The third approach to the problem of containi

fic emergency plans, to

ogical monitoring, where

g potential I y 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.

5

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
experime4ffords 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 levels. The first

class of experiments described in the guidelines are those which are

prohibited.

very serious hazard, should they in fact be hazardous. The prohibited

experiments include the following.

These are experiments which are judged to be of potentially

  (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 Control, of the United States Public Health

Service, or derives from cancer causing viruses classified by the National

Cancer Institute as moderate risk. Perhaps I should stress here that these

6

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.

or diphtheria toxin, and venoms from insects and snakes.

Examples of this class are botulinus toxin

(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 griculture.

(6) Finally experiments must be limited in scale to quantities of

flu d 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

7

not known to exchange genetic mater

and strictly defined containment is

ments it is mandatory to use biolog

certified by NIH as unlikely either

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.

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, n3t all recombinant DNA experiments yield

novel  combinations of UNA .... recombination between the DNAs of organisms

known to exchange genetic information in nature do not add uniquely man made

cells to She 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

ned, more stringent

many such experi-

hat have been

rigorously defined

Tnen the guidelines classify permissible

laboratory environments or to transfer the recombined Dl44 to other cells.

These agents  include certain derivatives of a bacterial species called g. II_ 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

biological containment.

The nature and manner of achieving biological containment with this

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

al in nature are recomb

required. Moreover, in

cally contained agents

to propagate outside of

8

i tse


cant


of a

   y in

   sma I


extens i ve

may be al

f,  it does not usually either establish itself or multiply signifi-

the normal bowel. These facts suggest that accidental injestion

number of bacteria by a laboratory worker would not result  in

spread of the bacterium outside the laboratory. The normal situation

ered 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.

combination used in a recombinant DNA experiment it will be necessary to assess

the possibilities  for transfer of the recombinant DNA  in order to eva uate 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.

e-

For any given -- E. coli K12 host-p asmid

The use of this bacteria has caused wide concern...and certain facts need

to be emphasized.  Only one strain of -7 E. coli, called K12,  is permitted by

the guidelines. Strain K12 is one of a large group of bacterial strains, all

of which are called 2. coli because they share certain properties  in common.

But they do no? all have identical properties. Some -I_ 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

9

whose principle successful ecological niche is in the laboratories of

molecular biologists and geneticists. It is not pathogenic. If it were,

you and I would not be here worrying about this research, since all the

molecular biologists would long since have disappeared. Pathogenicity s

a complex phenomenon .... dependent on several properties of the pathogen as

well as on the properties of the species being infected. It is very un ikely

that alterations in K12 brought about by insertion of recombined DNA will

make it into a pathogen .... but it is not impossible. It 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 L- 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 -__I E. coli, reservations about its use persist. It is important to

investigate alternative organisms ... and the Guidelines 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 4. 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,

10

such as might be invo

biological practice.

a moderate level of b

I eve


prov

They

host

ved in laboratory accidents, given standard micro-

Therefore, these existing systems, are judged to offer

ological 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

is called EK2. EK2 host-vector combinations must be demonstrated to

de a high level of biological containment by suitable laboratory tests.

are obtained by genetic modification of either existing E. coli K12

cells or the relevant vectors or both. More specifically, the guidelines

-_I

-II

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 (100 million). Various examples of the

types of necessary modifications are suggested in the guidelines.

-2 in

One additional level of contained E. a host-vector systems is defined

in the guidelines and is called EK3.

the specified containment properties have been demonstrated not only by

microbiological and genetic anaylsis but by appropriate tests in animals

i nc I ud i ng humans or pr i mates and other re I evant env i ronments.

EK3 systems are EK2 systems for which

  EK2 and EK3 host vector systems must be recommended for approval by

the decombinant DNA Program Advisory Committee and then certified by the

Director, NIH before use.

system must be submitted for consideration by the Committee.

several EK2 systems have been approved and certified. No EK3 systems have

Detailed data on the relevant properties of the

Thus far,

11

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, and a required biological containment

level, that is an EK level and the particular combination of the two reflects

the severity of

for the E. coli

DNA. In genera

relatedness of

--

the estimated potential hazard. The Guidelines are organized,

systems, according to the source and nature of the foreign

it is assumed that potential hazard increases with increasing

he 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 cell 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 cel

themselves are fragile and fastidious and there is little or no chance tha

a living cell could escape from a laboratory in the way that an -- E. coli

S

12

and whole human be

and the reports of

the early Salk pol

inoculated with it

the recipients

and SV40 cause


classified as

Because SV40 i

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,

ngs, as evidenced by the active production of antibodies

isolation of SV40 from humans. This virus contaminated

o vaccines and millions of people were inadvertently

in the middle 1950s. To date, there is no indication that

of the vaccine suffered any related difficulty. Both polyoma

tumor formation in newborn small laboratory mammals. They are

ow risk oncogenic viruses by the National Cancer Institute.

fects human beings, and also because SV40 and related viruses

have 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 pol yma.

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 llforeignll DNA fragment to be spliced onto the

viral DNA.

levels only.

Experiments with polyoma and SV40 are restricted to P3 and P4

The Guidelines also contain recommendations for experiments

plant cells will serve as hosts for recombinant DNA. The cells m

single plant cells grown under laboratory

parts, or small whole plants. This is in

guidelines address the question of recomb

organisms. Directions are given for modif

defining the roles and

assuring compliance wi

described, are primari

responsibilities of individua

h required containment levels

y directed at grantees of the

n which

ght be

conditions, or seedlings, plant

fact the only  instance where the

nant DNA experiments with whole

cation 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

s and institutions in

The procedures, as

Hat ional 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

s. Thus, the primary

o the guidelines is in

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 will

be used.  The application must  include certification as to the existence and

availability of appropriate facilities, procedures, and training. The

all of these must be carried out on a continuing bas

responsibility for conducting experiments according

his hands. Further, in applying for grants to carry

14

guidelines indicate that institutions in which recombinant DNA experiments

are carried out must establish biohazard committees which can serve to exam

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

scientific

in it i ated,

Guidelines


the invest


document.

ne

of the investigators judgment concerning the extent of potential  hazard and

the required containment would be by NIH study sections, during the normal

      review of the application. After approval, but before work is

                               tment to abide by the

understanding. Both

nstitution must sign this

the investigator must formalize his comm

by submitting to the NIH a memorandum of

gator and a responsible official of his

Failure, on the part o an investigator, to comply with the provisions

of the guidelines, may resu t  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.  All 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 agenc

whether ex i st i ng

to extend contro


as to how this m

es are involved. The Committee's charge was to determine

powers within the Federal regulatory agencies, are sufficient

to the non-federal sector....-to formulate recommendations

iy best be done, and to recommend legislation should that

be deemed necessary. The Committee has not yet completed its work, but I

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. It 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 F deral agencies may be expected to consult with their particular

constituenc es for advice on ways to proceed - the Department of Commerce with

interested

Agriculture with agricultural  interests and the NlH with scientific community.

ndustry - the Department of Labor with Unions, the Department of

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.