Protocols and Procedures


Biosafety Science
Estimate Hazard
Estimate Exposure
Risk Assessment and Characterization
Risk Management
Protocols and Procedures
EPA Risk Assessment Review Process

Infection followed by disease will depend on the microorganisms ability to multiply in the host and on the host's ability to resist or control the infection. It has proved useful to categories all microorganisms into 4 groups which define their pathogenicity to humans; only the first group are non-pathogens. This categorisation applies only to the infectivity towards humans, and is of significance only, therefore, for the contained use of organisms:

Hazard Group 1 Organisms that are most unlikely to cause human disease
Hazard Group 2 Organisms capable of causing human disease and which may be a hazard to laboratory workers, but are unlikely to spread to the community. Laboratory exposure rarely produces infection and effective prophylaxis or effective treatment is usually available
Hazard Group 3 Organisms that may cause severe human disease and present a serious hazard to laboratory workers. They may present a risk of spread to the community, but there is usually effective prophylaxis or treatment available
Hazard Group 4 Organisms that cause severe human disease and are a serious hazard to laboratory workers. They may present a high risk of spread to the community, and there is usually no effective prophylaxis or treatment

The intention of this categorisation, which applies to non-modified organisms as well, is to identify appropriate containment which would be required to protect those working with the organisms. The higher the hazard group, the greater the containment required to control the organism and ensure that it does not infect those working with it.

Pathogenicity is not a simple characteristic. Many genes must interact appropriately for a microbe to cause disease. the pathogen must possess and express characteristics such as recognition factors, adhesion ability, toxigenicity and resistance to host defence systems. Single gene modifications of organisms with no pathogenic potential or history, or even the introduction of multiple genes unlikely to confer pathogenicity are unlikely to result in unanticipated pathogenicity. For example, E. coli K12 has been disabled to remove some of the factors that might be associated with pathogenicity (wild type E. coli is a Hazard Group 2 pathogen). The factors which have been lost include the cell-surface K antigen, part of the LPS side chain, the adherence factor (fimbriae) that enable adherence to epithelial cells of human gut, resistance to lysis by complement and some resistance to phagocytosis. This variant of E. coli is a common host organism for genetic modifications within the laboratory.

The starting point for the risk assessment is, therefore, an assumption that the level of risk associated with the modified organism is at least as great as that of the host organism (until proved otherwise, either by direct observation, or by argument where the factors which are likely to enhance or decrease pathogenicity are considered as in the case of K12 above). Whether in the laboratory or in industry the capacity to choose a host means that in all but a few cases the host organism will have been chosen to be in Hazard Group 1. It is assumed that the modified organism will be used under the same containment as the host wild-type organism unless the modification inserts information which would alter the pathogenicity.

The vector has also to be considered, both for its own potential for pathogenicity and for its ability to transfer the insert to other than the intended organism -- horizontal transfer of the information. Most vectors used for E. coli contain no sequences which might result in pathogenic behaviour. The presence of genes coding for antibiotic resistance might be of concern. For most of these the antibiotic resistance is already common in the environment.

Most common E. coli vectors are transfer deficient, but the ability to transfer information either directly or with the assistance of other plasmids and the host range of the vector must be taken into account when considering the safety of the mechanism of insertion of the required genes into the host organism.

The properties of the insert are again of importance in considering the risk assessment for the modified organism. Clearly if the information encodes a toxic gene product, or one which is known to be likely to modify the pathogenicity of the organism into which it is inserted, the greater the risk. If the gene product is non-toxic and is not one which may pose a risk to the people working with the organism in containment, the risk management will largely be based on the pathogenicity of the host organism.

In most instances the characteristics of the donor organisms are of less relevance to the risk assessment than those of the host. If the donor organism is merely used as a source of well characterised DNA for a selectable phenotype or a promoter or other control sequence, the characteristics of the donor are unimportant to the risk assessment. If however, the insert contains genes which are biologically active, producing toxins or virulence factors, then information from the donor organism is of consequence. The construction of cDNA or genomic libraries make it essential to consider all the possible hazards associated with the donor organism, and in this instance, the hazard group may well have to be the higher of the two within which the host and donor fall.

It is now possible to examine the modified organism and consider the likely risk. During the 1970's Dr. Sidney Brenner and others in the United Kingdom attempted to systematise the approach by considering three factors -- Access, Damage, and Expression. The approach was incorporated in the United Kingdom's approach to risk assessment for contained use of bacteria, and is discussed in detail in a document produced by the Advisory Committee on Genetic Modification in the United Kingdom. The latest version of the guidance was published in 1999 and provides clear guidance as to the risk assessment for the contained use of genetically modified microorganisms (including any cells in culture). The guidance note is free and may be obtained from the Health & Safety Executive in Britain. More information is available by looking at the newsletters published by the ACGM which are available on the Internet on

Access is a measure of the probability that a modified micro-organism, or the DNA contained within it, will be able to enter the human body and survive there. It is a function of both host and vector. Depending on the organism being used, there are a number of routes of entry which allow access. The properties of the vector, particularly mobilisation functions need to be taken into account. In general if the organism is capable of colonising humans then access is high, whereas if the host is disabled so as to require the addition of specific nutrients not available in humans or outside of the culture media and is also sensitive to physical conditions or chemical agents present in humans, then the access factor is likely to be low.

Expression and Damage are usually associated with the insert and the gene product.

Expression is a measure of the anticipated or known level of expression of the inserted DNA; if the 'gene' inserted is intended to be expressed at a high level, for example, by deliberate in-frame insertion down-stream of a strong promoter, expression is likely to be high. If the insert is simply there to allow probes to detect the DNA, and is non-expressible DNA, i.e. with no foreseeable biological effect or gene containing introns which the host is incapable of processing, then the expression factor will be low. Examination of the final product, the modified organism itself, will determine the actual expression, which may be higher or lower than expected.

Damage is a measure of the likelihood of harm being caused to a person by exposure to the GEM, and is independent of either expression or access. It is associated with the known or suspected biological activity of the DNA or of the gene product. The activity of the organism which results in any toxic, allergenic or pathogenic effect need be taken into account within this parameter. It may be that the biological activity of a protein is dependent on the host cell system in which it is expressed. An oncogene expressed in a bacterium will have no discernible effect, when present in a human cell, problems may arise. The full biological function of many gene products require post-translational modification which will not occur within a bacterial cell normally. The potential biological activity of the gene product should be considered in the context of where an how it has been expressed and the effect on its structure and activity of the mode of manufacture. The 'damage' might be from

  • a toxic substance or pathogenic determinant that is likely to have a significant biological effect - damage is high
  • a biologically active substance which might have a deleterious effect if delivered to a target tissue
  • a biologically active substance which is very unlikely to have a deleterious effect or where it could not approach the normal body level. When cloning in E. coli the 'worst case' would be if all the E. coli in a person were replaced by the modified organism expressing a foreign polypeptide in an active form at a high rate. If all of these are absorbed in an active form and arrive at a site where they might have their maximum effect, what would be the damage?
  • a gene sequence where any biological effect is unlikely because of known properties of the protein or because of the high levels encountered in nature.

Once an estimate of each of these parameters has been made (in the United Kingdom this is numerical in steps of 10-3), they may be combined. The result provides a qualitative measure of the risk, and allows a containment level to be assigned for the use of the organism in order to protect those working with the GEM.

Unfortunately, this Brenner scheme is only easily applicable to a small class of experimental uses of modified micro-organisms, but the number of experiments in research laboratory environments which fit the requirements for the application of this scheme make its retention useful.

Modified organisms may be used in containment in laboratories (or pilot plants) or may be used in an industrial setting. It may be that the primary distinction here is not the size of plant or type of organism, but rather the skill and training of those working in the facility.

It is likely that a research or development laboratory will be working with organisms which pose a greater threat to either the individuals working therein or to the environment than do those organisms developed for large scale factory use. The great majority of organisms used in industrial production are well-characterised, 'familiar' organisms capable of being used under conditions of 'Good Industrial Large Scale Practice' or GILSP. Given that it is usually possible to 'choose' the parental organism into which a gene is inserted for a particular 'industrial' purpose, there would be no good reason to choose an organism likely to pose problems to either those working in the facility, or to the environment in the event of an escape.

The same logic would apply to the development stage where 'industrial' use of the modified organisms is being planned. There is a possible extra hazard in that it is at this stage that the modified genes may be inserted into the organism, and the unpredictability of insertion site may, arguably, require slightly greater care than that taken at the production facility.

In the research laboratory, organisms may be pathogenic to humans and/or to the environment, as it is here that fundamental research would be conducted. Experiments will involve organisms and /or inserts which may be injurious to the health of the workers or to those who are incidentally on site in the laboratory.

How are GMO's handled?

When the technology was first used, it involved the modification of organisms within a laboratory under very controlled conditions. The risks were perceived to be only to those working in the laboratory, and containment conditions were devised to attempt to ensure that the organism would not escape into the environment, or, if it should, it would have been designed so as not to survive in the open. This resulted in the assessment of risk only being associated with human health.

The technology has changed, and for the last ten years or more modified organisms have been used as biological factories within industrial environments. The volume of material may be considerably greater in the industrial or commercial environment than in the laboratory, and the individuals working with the organism may be less knowledgeable or competent at handling the organism. This implies that there is the possibility of accidental escape in a volume great enough for the modified organism to survive and persist in the open environment. There is also a risk of incidental release where waste from the industrial plant is not as carefully monitored or controlled as it would be in the laboratory. Hence in assessing the risks associated with industrial use of modified organism we have to take into account both the impact on human health (both for those working in the 'plant' and for those living close to it) and the possible environmental effects which may occur.

Modified organisms may be deliberately released into the environment. They may be crop plants which have been modified to change their characteristics or micro-organisms which are used, for example, for bioremediation on heavily polluted land. In both instances, the risk assessment which might be required would have to take into account the impact on the environment, which would include the health and safety of those humans living and working near the site of release.

Risk assessment is not an exact science. When a new characteristic is introduced into an organism, we are not absolutely certain of the site of introduction, and therefore of unrelated effects which may modify the organism in ways which we may not be looking for. The new gene or its products may interact in unexpected ways within our organism, or significantly alter the manner in which the organism interacts with its environment. As the risk when working with organisms in containment is largely restricted to considering the effect on human health and safety, the procedure may more readily be tabulated than when it is the environment that is considered the primary concern.

The first steps in risk assessment are to examine the host organisms, donor organisms, vector used for transfer of the gene, and the expected gene products.

    1. Where an organism has been used in containment for a very long time, and its characteristics have been described in detail, we are familiar with the organism. E. coli or Saccharomyces cerevisae are organisms about which a great deal is known. We know, for example, that no pathogenic strains of bakers' or brewers' yeast have ever been observed. These organisms are familiar. This familiarity allows some confidence in attempting to identify risks associated with their modification.
    2. The first presumption we are likely to make is that the modified organism is at least as hazardous as the host. For example, work with modified haemolytic streptococci will proceed in the laboratory in a similar way as with other streptoccoci of this type and known pathogenicity. However, more precautions are normally required for modified organisms as introduced external DNA might increase the hazard usually attached to these haemolytic streptococci. Formally such potential increase of the hazard is expressed by classification of the manipulated strain in higher risk category. The formulation "might increase" is important since it reflects the lack of our familiarity with the new strain. In some cases we shall observe the opposite - the new strain will be less invasive, the haemolysis less expressed, in short - the strain will represent lower hazard to human health. Nevertheless, since we cannot depend for sure on this in advance we are obliged to initially treat the new strain as more dangerous.

From this example we see that it is the absence of familiarity which brings the necessity of precautions when handling genetically modified organisms. This is also why we are asked to document our experiments and observations in more detail than working with common organisms. If any unexpected effect is observed in the later stages of an experiment careful documentation will make it possible to trace the experiment back and eventually come to the sources of the observed effect. On the other hand our detailed documentation will contribute to the building up of familiarity which in the future may result in amendment of the risk assessment and assignment of a lower level of containment than that initially assigned.

What is the essence of precautions required by regulations for contained use? The use of the GMO should be "contained". The containment could be physical, where there are real barriers to prevent escape, or biological, where the organism is designed not to be able to survive in any environment other than that of the laboratory. Physical containment means that we are asked to keep the GMO within barriers which prevent its escape from the designed space. In this way the GMO will be under control. Such barriers are usually represented by walls, fences, boxes, filters and other mechanical constructions safely preventing the GMO from escaping

Barriers may be also of a chemical nature. Solution of phenol or hydrochloric acid will prevent bacteria from invading the environment. Also heat is used in many systems, e.g. fermentors. Certain principles can help to improve containment but single factors are not sufficient to fulfil the conditions of containment. Laminar flow, negative air pressure and in many cases also so called biological barriers fall in this category.

"Biological barriers" need careful consideration. Let us have a strain of bacteria representing a risk to the environment which is not able to synthesise lysine and folic acid. Consequently it will grow only in media supplemented with these two growth factors. Can we consider this deficiency as a "biological barrier" which will prevent survival and spreading of this strain when it escapes from containment? In practice, are we allowed to pour a culture of such strain in the drain? The answer is no. Sewage water contains great selection of organic compounds many of them can be considered as "growth factors" for auxotrophic bacteria. Let us have another example. Can we open the door of a stable and let the transgenic cow go free for a pasture on the meadow? Certainly we can. There is no possibility that the gene introduced in the genome of the cow will escape from the animal and will contaminate the environment. (but only if it is the female!)

These examples bring us to releases of modified organisms. In general we can call "release" any removal of the GMO from physical barriers which limited its presence within a closed space. This could be accidental 'escape' or intentional action. When we start using GMO's we must think about the possibility that such accident may occur, think of the likely consequences of the escape, and if necessary, prepare steps which have to be taken.

When considering deliberate release it is clear that the risks will differ depending on the organism released. Modifying an animal virus so that is capable of binding to human receptors is likely to pose significant risk to the human population, and a risk assessment would normally indicate that this should not be allowed. We have already indicated that the release of a cow into a field constitutes (effectively) no risk. Ten years ago a company in the USA was in serious trouble when their researchers placed pots with several seedlings of a transgenic tree on the flat roof of the building without the permission of the authorities. To-day such an experiment would be classified as of little risk, particularly if the plants were not allowed to produce pollen. Only a burglar stealing the pots or a wind of an tornado strength might spread the modified genome of these trees in the environment. It is only recently that permission for field application of transgenic microorganisms introduced into soil has been given. At the current stage of knowledge we are not able to predict nor to monitor the transmission of a gene in the community of native soil microorganisms. Therefore nobody has been willing to step in the dark. Again we see how important is our familiarity with traits and behaviour of parent organisms and extend that to those which result from gene engineering techniques.

Plants require special handling. As soon as they form pollen their genome can be transferred to other plants of the same or close species. Since this transfer may occur through different vectors - wind, insects, man, water - measures ought to be taken to eliminate the possibility. Examples include the use of "male-sterile" varieties which does not form pollen. Alternatively, flowers may removed before the pollen is formed, or the flowers could be 'bagged' to ensure that pollen cannot escape. This is not normally possible in large-scale field experiments. We may attempt to ensure that sexually compatible plants are a considerable distance away from those genetically modified so that the transfer of pollen is unlikely. In such cases the "safe zone" around the field should be kept free of relatives which could be pollinated by the transgenic cultivar. The size of this zone could be assessed from the largest possible radius the pollinating insects (e.g. honey bees) can travel. In the case of wind pollination this distance could be very large, unless the pollen is viable for a relatively short time. Many experiments have been performed to attempt to identify 'isolation zones' for particular crop species.

In general the handling of GMOs is dominated by two precautions: to protect the health and safety of people who have a direct interaction with the GMO (laboratory workers, factory workers, cleaners in the laboratory) and to protect the environment which will include people, water, earth, air.

Last Modified: May 21, 2001
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