Preamble

The first uses to which genetic modification were able to be put involved the use of micro-organisms in containment. Almost from the very beginning of the use of recombinant techniques it was realised that organisms could be designed which might prove to be 'dangerous' to those working with them, and to the environment. It was recognised that the technology provided a new means of forming organisms which were significantly different from those that had been known up to that point.

Because the first uses of modified micro-organisms were in the laboratory, it was primarily human health and safety that mattered. It is presumed that biological agents are hazardous, and may affect those handling them in predictable ways.

Containment must be used when micro-organisms are used whose function is known to be hazardous to humans, or where the presence or function of the organisms is unknown (as in clinical samples). Where an organism is not known to have pathogenic properties, it is assumed to be safe, presumably because had it been pathogenic (to immuno-competent individuals) it would have been observed to be so. Most human pathogens have been described and assigned by the WHO to 4 groups which relate to their likely effect on anyone coming into contact with them. Problems arise when considering new organisms created in the laboratory. How may these be assigned?. Where can a classification of risk to those in the laboratory or development environment be obtained?

• Group 1 biological agent means one that is unlikely to cause human disease;

• Group 2 biological agent means one that can cause human disease and might be a hazard to workers; it is unlikely to spread to the community; there is usually effective prophylaxis or treatment available;

• Group 3 biological agent means one that can cause severe human disease and present a serious hazard to workers; it may present a risk of spreading to the community, but there is usually effective prophylaxis or treatment available;

• Group 4 biological agent means one that causes severe human disease and is a serious hazard to workers; it may present a high risk of spreading to the community; there is usually no effective prophylaxis or treatment available.

The presumption that the organism is a micro-organism results from the presumption that it is the health and safety of those who come into contact with the organism that need protection. Having identified the Group into which an organism falls, it is possible to identify the containment requirements that are needed to protect the handlers.

The initial concern for the health and safety of the handlers must now be modified to take into account the impact on the environment in the event of escape from containment or where release is intended. A plant or animal pathogen may have no effect on humans, is likely to be placed in Group 1, and minimal containment requirements identified. If this increases the probability of escape into the environment, problems may well arise. If environmental risks are taken into account, then it is not only micro-organisms that fall within the terms of any containment requirements, for animals and plants may have implications as well.

A consideration of the safe use of genetically modified organisms, whether for use under contained conditions or for releases into the environment is hard. We really do not know where to start, particularly for release. We know that the introduction of novel species into a new environment can (and does) go wrong on occasion. Does the insertion of a gene into an organism that has been present in an environment for a very long period of time make a new novel organism? Where do we start assessing the risk associated with the insertion of the gene into a well-characterised organism, let alone those which might be novel in a particular environment? What happens when we agree that a particular organism is safe in Britain or in the Netherlands (for example), does that mean it is completely safe when used or grown in Spain or Greece? If designed for safe use in a temperate climate, is the organism safe for 'escape' into a tropical environment? If not, how do we assure that the permission to proceed to the use of the modified organism in one country (or region) does not extend to the whole world? This problem is exacerbated by the rules which have been put into place through the operation of the World Trade Organisation, which would appear to require an importing country to provide scientific evidence for the lack of safety of a particular import rather than require the exporting country to justify its safety assessment.

The risks posed by the newly manufactured organism are dependent on the uses to which they are put, but significantly often the risks may be considered in a narrow framework simply because of the particular use, and perhaps an extension of use of the organism may not result in a 'proper' reconsideration of risk; in examining the safety of the organisms we have to try and determine what should be considered hazardous and it has not proved simple to identify the issues contributing to risk. Let me try to use an example to explain this problem. When a field trial of a new genetically modified oil-seed rape is proposed to the UK Government, the proposal will not usually consider the use of the plant as a food or feed, for the proposal is only concerned with the initial field trials. The ability to transfer the genes to wild relatives or to other oil rape plants adjacent to the trial field has been one of the main concerns, although whether this should be of concern is open to debate in the UK. Where seed is kept from year to year it could be a major problem, but where new seed is purchased in each year, and the seed merchant guarantees the quality of the seed, is there a problem if a gene for say herbicide resistance is passed into wild relatives?- only where the wild relatives constitute weeds and need controlling within the agricultural environment itself, not if they only grow in areas that are not controlled by herbicide application. But there is a reason for trying this new construct, which must be its commercial exploitation. Should we not consider the general implications of the use of this new organism at an early stage, rather than waiting and considering the safe use of the organism within the narrow confines of the application itself.

At the other end of the scale, and one that has caused considerable problems to Europe over the last few months, and which will continue to provide real problems, is as follows. Suppose we approve an organism as completely safe for use within the European Union, whether it is anywhere in the geographical confines of Europe or restricted to 'cold' climate agriculture (for example). We have made this product and obviously want to sell it as widely as possible -They have their own legislation relating to the use of such organisms, and may not be happy with our safety assessment; can they stop us marketing our 'product' because of safety concerns? What weight should our safety assessment play in their consideration of the safety issues? Our permission to market the product may be just as valid as their decision not to allow marketing of the product, perhaps because of indigenous organisms. How do we guard against protectionism cloaked within safety concerns? In the case of a developing country, where there might not be legislation that governs biosafety. Should we simply export to them without considering the risks that our organism might pose to them? If so, are we prepared to face the risk that a slightly 'more' modified organism returns to haunt us?

What about the opposite problem, where we receive biological products from other countries. We may know that it has been modified if it has come from the USA or Canada, but if it comes from countries which have no legislation in place to allow for the assessment of risk associated with modified organisms? How likely is it that their products will appear on our markets and pose problems for us in the future.

Within the European Union there are two directives that cover this part of Biosafety. Both directives were drafted in 1990, and required implementation within member states by 1993. 90/219 governs the contained use of genetically modified micro-organisms, whilst 90/220 governs both the release of the organisms into the environment and the marketing of any modified organism, whether for release of for contained use. The primary difference is that 90/219 assumes containment, and that the regulatory structure can be specific for the member state. 90/219 sets minimum standards for the making, use or keeping of the organisms, and member states are free to have legislation that extends both the range and scope of the directive. In the UK the legislation covers all organisms, not only micro-organisms. Release is different, for even an experimental release has the potential for crossing the boundaries between states, and therefore, the harmonisation of community legislation is important. The marketing of modified organisms is also likely to be community-wide, even though geographical constraints may be applied. 90/219 is therefore global, and must be implemented 'as is' within the community, without allowing real discretion in member countries.

These directives have been criticised by many, for a multitude of reasons: perhaps the most important reason (not usually stated) is that they appear to be different from that which has been implemented in the USA. There are major problems with both directives.

It was always intended that the directives would change with time, in that the authors wrote into it that product specific legislation could exempt a particular substance from consideration as long as the risk assessment procedure in the specific legislation was at least as comprehensive as that identified in 90/220. It was also assumed that it was based on a presumption of a learning curve, that each case would be argued on its merits, but taking into account evidence and information obtained from previous releases of similarly modified organisms. Hence the legislation allowed for the institution of simplified procedures - or 'fast-track', where consent to release the modified organisms could be given much more quickly and easily than for a totally unknown construct. There are therefore, no major changes expected in this Directive, as most desirable changes (as far as industry is concerned) can be accomplished by product-specific legislation or through fast-track procedures. We remain different from the United States in many important respects.

Are there differences in the way in which containment is handled in different countries?

There are significant differences in the containment requirements in different countries. Although both the United States and the United Kingdom developed their regulatory systems following the Moratorium self-imposed by scientists in the 1970's, the regulatory systems moved apart significantly. In the United States it was decided not to impose new regulatory burdens on the biotechnology laboratory or industry. Guidelines for the safe use of modified organisms were introduced, in particular the NIH Guidelines, which imposed a set of safety precautions on those funded by the NIH for the safe use of modified organisms. Most industries, although not funded by NIH, followed these guidelines, at least in spirit.

In Europe, however, a statutory regulatory system is in force, which requires an assessment of the risks associated with the use of modified organisms in containment. The statutory system in Europe also depends on a Directive (90/679) which defines the conditions in which biological agents (whether genetically modified or not) may be used in the workplace. Most of this discussion relates to the system in place in Europe, as it is not dissimilar to that expected but not required by law in many countries.

Containment

We turn to the use of genetically modified organisms in containment. Containment includes both the research stage, where modifications are made, development work whether in the laboratory, greenhouse or growth room, and in plant where modified micro-organisms are used as factories to produce new products. In this instance it is not intended that the modified organism is released into the environment, and if being used at a commercial or industrial level, they would only be being used as factories. The concerns are different. There is a peripheral problem with the environment, only if there is inadvertent escape, or if there are waste products that might contain viable organisms does the environment need considering. The true concern is with human health and safety - protecting all those who might come into contact with the organisms during their production or use. The approach taken in the risk assessment is different to; here we devise confinement and containment that will minimise a risk. The risk might be substantial, like modifying HIV in order to unravel its 'secrets', or with polio virus to try and understand how it works, or why it only infects particular types of cell. The risk management procedures will minimise the risks and allow the work to proceed. How again, are we to determine the risk?

What is protected when considering contained use?

• the health and safety of the human on which the procedure is applied (if any)
• the health and safety of workers in the laboratory or industry in which the organisms are handled, used, made, kept, etc.
• the health and safety of others who have access to the workplace, or near it (cleaners, secretaries, students, visitors)
• the health and safety of all other persons who might come into contact with the organism if it is incidentally or accidentally released into a wider environment.
• the health and safety of animals within the laboratory environment if they are susceptible to the organism, whether they are used as 'guinea pigs' for testing of the modified organism, or if infection is unintended
• the environment of the accidental or incidental release of the modified organism.

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Europe

The original Directive which was produced for the European Union, 90/219 was clearly flawed. It worked on the basis type of work and of the hazard, rather than on the true risk.

The Directive defined two types of activity, Groups A and B. If the work was for research or development purposes, or non-commercial, or non-industrial, it was group A, otherwise B. The only purpose of these two groups was to identify the notification and consent requirements for the work. The separation is at least partially justified on the basis of the risk to workers - Group B work might involve less qualified or involved personnel who would not understand the risk, but takes little account of either risk or hazard.

Micro-organisms are then classified as type 1 or type 2. Type 1 organisms are those that pose no risk to human health or safety; type 2 organisms are all others. The classification is largely based on the host organism. I find it difficult to link the risk assessment with these classifications, which simply identify the needs of notification and consent once again. A risk assessment is still needed, but that which is closely defined is not the risk, but whether a consent is required.

Discussions concerning this Directive and possible modification have proceeded almost since it was first agreed in 1990. A change to a risk rather than hazard based system, with notification and consent based on risk rather than hazard or type of activity has been thought to be important, but it is not easy to implement these ideas. Some member states believe that the current Directive is working well, others would prefer a more logical approach.

This Directive is undergoing radical modification and a version has been sent to the European Parliament. The Commission is working on a draft that I have provided with this talk, and it demonstrates a complete change in philosophy, for it identifies the containment needed to minimise risk and links the risk assessment - risk management system with the consent - notification system. That which I have to say here concerns the latest version that I have seen, somewhat later than that presented to the parliament, and dated November 1996. I have to state that I believe the draft Directive to be a massive improvement on that which we have now, and workable in that it directly relates the results of the risk assessment to the containment that would be required to minimise the risk.

The main provision is within article 5 of the draft, which states

1. That the reason for the directive is primarily to require Member States to ensure "that appropriate measures are taken to avoid adverse effects on human health and the environment that might arise from the contained use of genetically modified organisms"
2. The user shall carry out an assessment of the contained uses as regards to the risks to human health and the environment that they may incur.
3. The assessment shall result in a classification of the contained uses in four classes applying the procedures set out in Annex III of the directive, which will result in an assignment of containment
   1. Class 1: Activities of no or negligible risk that is activities for which level 1 containment is appropriate to protect human health as well as the environment.
   2. Class 2: activities of low risk that is activities for which level 2 containment is appropriate to protect human health as well as the environment.
   3. Class 3: activities of moderate risk that is activities for which level 3 containment is appropriate to protect human health as well as the environment.
   4. Class 4: activities of high risk that is activities for which level 4 containment is appropriate to protect human health as well as the environment.

The directive continues to define the requirements for notification or permission for each of the 4 classes defined above.

The major requirements of the Directive apply to the risk assessment and to the definition of techniques that are included within the definitions. These both appear in annexes, and there is still discussion as to which of these annexes should be adaptable to technical progress (modifiable without having to modify the directive) and which not. The discussion that follows closely follows the text of the draft directive, for obvious reasons. The discussion between competent authorities and the commission to try to achieve an agreed text to this directive often becomes bogged down in detail, which results in extremely careful choice of words. As the directives are translated into each of the Community languages, and the meaning of words in each of these languages may well differ, this seems pointless, but problems between member States may often be resolved by judicious choice of words.

In Annex III we see the Principles which would be followed for the assessment of risk, which include

1. A definition of harmful effects (including allergenicity and toxigenicity, disease, establishment or dissemination in the environment, gene transfer)
2. Identification of potential harmful effects, particularly those associated with the recipient or host organism, the donated material, the vector, donor if used during the operation rather than just its sequence, and the resulting modified organisms.
3. the characteristics of the activity
4. The severity of the potentially harmful effects (hazard?)
5. The likelihood of these potentially harmful effects being realised.

The procedure used to perform this risk assessment involves first identifying the harmful properties of the host organism, and then the properties of the donor organism, vector and finally the modified organism, making sure that only organisms for which

• Both donor and recipient are unlikely to harm humans, animals or plants (where appropriate)
• the vector and the insert are such as not to provide a phenotype likely to cause disease to humans, animals or plants in the facility in which the organisms are used, or to cause adverse effects in the environment;
• The modified organism is unlikely to cause human, animal or plant disease or to harm the environment.

are classified as class 1.

The user should take account of the various tables classifying pathogens for animals, plants and humans (90/679 and 93/88) in assigning organisms to classes.

Selection of containment and control measures should then be made on the basis of the level of risk associated with the modified organisms together with information on the likely environment into which the organism could escape; characteristics of the use (e.g. scale) and any non-standard operations. This should lead to assignment to one of the classes defined previously.

A variety of tables appear in the draft directive to define the conditions of containment that are needed for each of the four classes of risk.

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Risk Assessment and Risk Management.

1. The legal process which regulates the use of genetically modified organisms is concerned with the health and safety of those in the workplace and with the possible harm that may be caused to the environment by the modified organism. A formal risk assessment is required to identify any likely risks and management procedures must be designed to minimise them
2. Risk Assessment depends on an assessment of the hazard associated with the modified organisms or the procedures used in handling the organism, and on the likelihood of the hazard occurring.
3. For CONTAINED USE, where it is presumed there are both physical and biological barriers which limit the likely impact on the environment, it is almost solely the health and safety of those who are likely to enter the laboratory or factory that causes concern. The assessment of risk considers the likely effects of the modified organism on the humans using it, including those who enter the facility but are not aware of the sort of work being done (cleaners, secretaries...). It must also take into account both the incidental release of the organisms into a wider environment (waste streams, rubbish) and accidental release - what are the likely effects if the organism escapes from its physical containment, not only on human health and safety outside the containment facility but also on the environment?

This is crucially important where the 'micro-organism' being used is a plant or animal pathogen, or even a plant or animal cell. The initial test, for risk to humans, indicates lack of problem and hence safe usage in relatively light containment, but the impact on the environment may be severe should they escape, and hence the need for greater containment (both physical and biological) is indicated.

1. Where the organism in containment is an animal or plant, the risk to the environment on escape is the most crucial, although risk to humans remains a consideration. If 'animals' are mentioned, it is often assumed that large farm or domestic animals - cats, dogs, sheep, pigs or cows are involved. It is these animals where the risk is probably least, and to some extent, biologically contained (There is little risk of genes inserted into a cow 'escaping' into the environment, but there is risk associated with the escape of such genes from a tom-cat). Modified animals which might pose a real threat to humans might be mites or insects and a consideration of the likely effects of escape or infestation becomes more important as the biological containment is less effective.
2. Where the organism constitutes a risk to public health, special considerations apply. A report recently published by the Advisory Committee on Dangerous Pathogens in the UK on Microbiological Risk Assessment tabulates a risk assessment procedure as follows for risk assessment, not specifically for genetically modified organisms, but in general (summarised): A United States Presidential and Congressional Commission has recently reported in similar terms:

• A statement of why a risk analysis is needed - the cause of concern. This may include questions relating to uncertainties that need resolution

• Risk Assessment - (i) identification of the source of the hazard and the conditions under which adverse consequences could occur; and (ii) reviewing and quantifying the risk consequent on each hazard. The consequences of failure to identify a serious hazard may be grave. The procedure would involve
  • Assembly of available information from relevant data sources
  • Definition of the hazard and its potential harm (qualitatively expressed), explicitly stating any essential assumptions made and the circumstances in which its harm may be expected. This definition might include consideration of the life cycle of the organism, route of transmission, susceptibility to infection, available treatment or prophylaxis, consequences of infection...

• Risk Importance - a judgement of the significance of the risk and the probability of the hazard being expressed. Information which might be available includes experimental evidence, epidemiological information, predictive modelling, information on pathogenic mechanism, route and consequences of infection, susceptibility of the population and the natural history of the disease.

• Production of a formal record - a clear; comprehensive and concise record should be accompanied by a summary of the data on which the assessment was made and an appraisal of its quality.

• Testing of the robustness of the scenario of the risk assessment - to challenge the susceptibility of the outcomes of the assessment to changes or errors in the data and assumptions on which the assessment was made.

• Risk Management - the decision on and implementation of action to eliminate or minimise risks.

• Risk Communication - the communication of information on the risk and on the decisions made to minimise or combat it

• Risk Monitoring - the assessment of the effectiveness of control measures.

1. Where the organism is DELIBERATELY RELEASED INTO THE ENVIRONMENT, or where the physical containment is not likely to be completely effective, the environmental effects of the organism become more important, and a detailed assessment of the risk to the environment is needed.
2. Risk assessment identifies the hazards which might be encountered, and the probability of the hazards being realised. Risk management techniques are used to modify the probability of the hazard being realised so that the risk is minimised. In some cases this might require significant changes to the design of the system. This process is effectively a filter system, which may allow an undesirable product through.
3. Whether for contained use or for deliberate release the hazard identification concentrates on four factors, which will be discussed in some detail in the chapter on risk assessment methodology:

• the genetic alteration
• the phenotype of the wild-type host and donor organisms, and of the vector used to transfer the information
• the phenotype of the modified organism
• the specific environment into which the organism is to be placed.

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How are GMO's handled?

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.

1. 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.
2. 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
3. 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.
4. "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!)
5. 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.
6. 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 in order to test the effect of weather. 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.
7. 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.
8. 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

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Risk Assessment for the contained use of modified Organisms

It is easiest to consider the risk if we start assuming containment, for all procedures which finally result in a marketed modified organism will begin in the laboratory. In some senses this is where most of the hazard is likely to be found, or where risk potential is greatest, for the least is known about modified organisms in the research environment, and scientists are more likely to modify pathogens or try and identify genes which will be 'useful' from a variety of organisms which are not fully characterised.

If the organisms are contained we have first to consider only the probable effect on human health and assume that our containment system will isolate our organism from the environment.

The European Union defines "contained use" as being within physical barriers, whether or not supplemented by chemical or biological barriers to escape or release into the open environment. The definition includes the modification of organisms and their storage, culture, use, transportation or destruction for which physical barriers are used to limit their contact with both the general population outside the containment and the environment. A genetically modified large animal might be considered to be in containment, if the definition depended on an ability to recall the 'organism' in the event of a problem, rather than a physical fence between that organism and the wider environment.

In order to assess the risk for contained use, it is usual to follow a scheme where we

1. consider the predicted properties of the genetically modified micro-organism to determine if there are any potential mechanisms by which it could represent a hazard to human health.
2. consider the likelihood that the genetically modified micro-organism could actually cause harm to human health
3. assign the containment which would be necessary to safeguard human health
4. identify any hazards to the environment and assign any additional containment measures to assure that the environment is not placed at risk.

Our modified organism is conceptually separable into the host organism, into which genetic information is inserted; the donor organism, from which the genetic information has been derived; the vector which shuttles the information between these organisms, and the insert, which contains one or more genes which display biological activity. It is useful to consider each of these in attempting to assess the likely hazard posed by the modified organism.

When working in containment all cells, whether they are microorganisms, plant, animal or human cells, are considered to be micro-organisms when used in culture. This is only important in that the European Community Directive (EC 90/219) deals with contained use of modified micro-organisms. Animals and whole plants used in containment are not covered by a Europe-wide Directive, although many of the countries have "over-implemented" the directive so as to include all organisms used in containment.

In general, however, it is only microorganisms which are considered pathogenic to humans, although plant cells may produce toxic and allergenic substances which pose a hazard to the worker in the containment facility. (The concept of toxicity includes mutagenicity, carcinogenicity, neurotoxicity and environmental effects).

For each of the donor, host, vector and modified organisms we may consider the hazard they pose, which will provide information which allows a first approximation to the hazard likely to be posed by the modified organism.

Before considering the properties of each of these, we may consider whether it is likely that the modified organism differs from the host organism at all. It is almost certain that the genetic information transferred to the host cell is an infinitesimal fraction of that incorporated within the host cell. The gene or genes that are inserted are likely to be well characterised and the changes in phenotype are predictable - otherwise there would be no point in the modification (except in the laboratory where making gene libraries provide the possibility of totally unknown mixtures of organisms). However, the genetic modification might affect the host range of the host organism, or its capacity to utilise a different set of metabolites, or might convert the host organism into a pathogen, or alter its ecological niche, or the balance of organisms within that niche. The point of insertion of the characterised genes within the genome of the modified organism is unknown. All that this paragraph says, however, is that we should treat our organism as a totally new organism, and base a risk assessment on encountering an unknown organism. Are the hazards unique to modified organisms, or should we treat this as equivalent to a newly isolated organism?

If we were to treat this as a newly isolated organism, we would be discarding a great deal of information which we possess! Where would risk-assessment start? The implication would be that the organism should be treated as a very dangerous pathogen until proved otherwise, and surely the insertion of a non-toxic gene into a species like Saccharomyces cerevisae which has never been shown to have pathogenic properties would be extremely unlikely to produce a very dangerous human pathogen.

We obviously start with information relating to the host species, take into account all the information available from the donor and vector to allow a preliminary risk assessment for the final modified organism. The risk management procedures that will then follow will include some monitoring to ensure that the risk assessment performed in this way is not seriously wrong.

The genetic modification might affect the host range of the host organism, its capacity to survive under different conditions, and the susceptibility to effective treatment or prophylaxis in the event of infection. It might also alter its capacity to utilise different substrates or alter its balance with other ecologically interrelated populations. Nevertheless, it is the pathogenic properties of the host organism that determine the starting point for our assessment of the likely hazard posed by the modified organism.

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 categorise all microorganisms into 4 the four WHO 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:

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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 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 category 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, but for most of these the antibiotic resistance is already so common in the environment that it can be discounted.

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 great 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, toxins or virulence factors, then information from the donor organism are 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 or 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 1993 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 http://www.shef.ac.uk/~doe

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.

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Damage and expression 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 GENETICALLY MODIFIED MICRO-ORGANISM, 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 range of '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? In the case of insulin, this perturbation is unlikely to be above 10% of the normal insulin levels, and therefore the damage is not likely to be great.
• 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 GENETICALLY MODIFIED MICRO-ORGANISM.

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.

Level 1 is the lowest level or containment, and requires no extra precautions above those required for good microbiological practice. In general, this means that

• Laboratory personnel must be instructed in the procedures used in the laboratory.
• The laboratory should be easy to clean. Bench surfaces should be impervious to water and be resistant to acids, alkalis, solvents and disinfectants.
• If the laboratory is mechanically ventilated, an inward air flow into the laboratory should be maintained by extracting room air to the atmosphere.
• The laboratory must contain a wash handbasin or sink that can be used for hand washing.
• The laboratory door should be closed when work is in progress
• Laboratory coats or gowns should be worn in the laboratory and removed when leaving the laboratory suite.
• Eating, chewing, drinking, smoking, storing of food and applying cosmetics must not take place in the laboratory.
• Mouth pipetting must not take place
• Hands must be disinfected or washed immediately when contamination is suspected, after handling viable materials and also before leaving the laboratory.
• All procedures must be performed so as to minimise the production of aerosols.
• Effective disinfectants must be available for immediate use in the event of spillage.
• Bench tops should be cleaned after use
• Used laboratory glassware and other materials awaiting disinfection must be stored in a safe manner. Pipettes, if placed in disinfectant, must be totally immersed.
• All waste material which is not to be incinerated should be rendered non-viable before disposal.
• Materials for disposal must be transported without spillage in robust containers
• All accidents and incidents must be recorded.

The conditions for higher levels of containment, where the organism is considered a pathogen, are listed in the guidance note (see footnote ). For level 2, the major addition are the need to ensure that access to the laboratory is restricted to those needing to enter; that there be adequate space for each worker (at least 24 m³); an autoclave must be readily accessible and all waste materials must be made safe before disposal either by autoclaving or by incineration.

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Table 1:

Summary of Laboratory Containment Requirements

	Level 1	Level 2	Level 3	Level 4
Laboratory suite: isolation	No	No	Partial	Yes
Laboratory: sealable for fumigation	No	No	Yes	Yes
Ventilationinward airflow/ negative pressure	Optional	Optional	Yes	Yes
through safety cabinet	No	Optional	Optional	No
mechanical: direct	No	No	Optional	Yes
mechanical: independent ducting	No	No	Optional	Yes
Airlock	No	No	Optional	Yes
with shower	No	No	No	Yes
Wash handbasin	Yes	Yes	Yes	Yes
Effluent treatment	No	No	No	Yes
Autoclaveon site	No	No	No	No
in suite	No	Yes	Yes	No
in lab: free-standing	No	No	Optional	No
in lab: double-ended	No	No	No	Yes
Microbiological safety cabinet / enclosure	No	Optional	Yes	Yes
Class of cabinet / enclosure	-	Class I	Class I/III	Class III

There will be risks to the environment for contained use of modified organisms as well. The escape of modified organisms used in laboratories should be of little significance as they should have been disabled so that, in the event of escape, they will be unable to survive. However, the assumption has been that the only concern is risk to human health and safety. The risk assessment has been predicated on this, and the possibility that the organism may be a danger to other organisms within the environment has not been fully considered in the discussion. The concept of the 'environment' which includes land, air and water as well as other organisms and humans, is so broad when compared to the enclosed environment of the laboratory that is it difficult to define a clear step-wise approach to risk assessment. The risk assessment must try to consider all possibilities of what could go wrong, and attempt to ensure that these cannot happen, largely through the design of the organism being used. Methods for retrieving the situation should an organism escape from containment become important, and need to be planned at the outset rather than relying on the containment procedures to work. This is particularly important where the organism is a Level 1 organism as it will not infect humans, but if it escapes could be disastrous to plants, insects or other animals.

The extra hazards that need be taken into account so as to assess the risk should an organism escape from the laboratory environment include (a great deal of that which follows was included in Chapter 1, but is felt to be important enough to be repeated here):

The potential to survive, establish and disseminate within an environment distant from that in the laboratory. This may include the displacement of other organisms, or the modification of the ecosystem so that other organisms disappear or are replaced. Indirect effects may follow the establishment of a new organism.

Pathogenicity to animals and plants is clearly significant. The characteristics of the host organism and the modified organism which are relevant to pathogenicity, toxicity, virulence, allergenicity, colonisation, predation, parasitism, symbiosis and competition need be considered. If the host is pathogenic, the modified organism may be to a greater or lesser extent, and this should be considered.

The potential for transfer of the genetic material should also be considered. Conjugative plasmids, transmissible vectors or transposable elements which could contribute to the undesirable spread of genetic material between the GENETICALLY MODIFIED MICRO-ORGANISM and other organisms must be considered in the risk assessment.

The products of the gene expression which might be toxic to organisms other than humans needs consideration, or where the precautions taken in the laboratory to protect humans from the toxic effects are no longer present in the environment. An organism that has the potential to cause negative effects on other organisms as a result of an inserted gene coding for a toxic product will pose a hazard.

The organism may have the potential to cause negative effects on other organisms and these should be considered.

The loss of a gene in the organism is not a hazard in itself, but such instability may lead to the incorporation of the genes in other organisms which may result in harm to the environment. Again this must be considered, even though escape is not expected.

Large scale use of modified organisms in containment is different from use in the laboratory in a number of ways. In the first instance, it is almost certainly true that the organisms used in development or for industrial and commercial use are non-pathogenic. They are generally used under conditions of 'Good Industrial Large Scale Practice' (GILSP) defined by a working group of OECD.

The hazards posed by large-scale fermentation of genetically modified micro-organisms are of the same nature as for other biological agents, in particular

• infection hazards - the potential for disease following exposure to the organism;
• toxic, allergenic or other biological effects of the non-viable organisms or cell, its components or its naturally occurring metabolic products;
• toxic, allergenic or other biological effect of the product expressed by the organism

There is nothing intrinsically more hazardous about the large scale use of genetically modified organisms in containment other than the potential for a greater degree of exposure to an organism and its biologically active products or the possibility that workers in an industrial plant are less skilled at handling biological material than laboratory workers. In general, large scale users may well have chosen the best characterised host organism, as knowing the conditions under which the organism is likely to thrive makes industrial use more cost-effective.

The criteria for organisms to be used under Good Industrial Large Scale Practice conditions include

• The host organism must be non-pathogenic to humans with no adventitious agents and an extended history of safe industrial use, or there must be environmental limitations permitting optimal growth in the industrial setting but limited survival without adverse consequences in the environment
• The modified organism must also be non-pathogenic and as safe in the industrial setting as the host organism, but with limited survival without adverse consequences should it be released either accidentally or inadvertently into the environment.
• The Vector or insert must be well characterised and free from known harmful sequences; it should be limited in size as much as possible to the DNA required for the intended function; should not increase the stability of the construct in the environment (unless that is part of the design of the organism). It should not transfer any resistance markers, particularly to microorganisms not known to acquire them naturally.

Organisms which are used to manufacture biologically active chemicals will obviously not fall within the definition of GILSP in many circumstances

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References

1. Contained use is defined the new article 2 to be any activity in which micro-organism are genetically modified or in which such genetically modified micro-organisms are cultured, stored, transported, destroyed, disposed of or used in any other way, and for which specific containment measures are used to limit their contact with the general population and the environment, and micro-organism means any microbiological entity, cellular or non-cellular, capable of replication or of transferring genetic material including viruses, viroids, animal and plant cells in culture.

2. Microbiological Risk Assessment: an Interim Report, 1996, Advisory Committee on Dangerous Pathogens, HMSO London.

3. Grinstead J (1995) "Risk Assessment and Contained Use of Genetically Modified Microorganisms", in Genetically Modified Organisms: A Guide to Biosafety, Tzotzos GT (Ed) CAB International, Oxford, ISBN 0 85198 972 1

4. Grinstead J (1995) "Risk Assessment and Contained Use of Genetically Modified Microorganisms", in Genetically Modified Organisms: A Guide to Biosafety, Tzotzos GT (Ed) CAB International, Oxford, ISBN 0 85198 972 1, p26-27.

5. Grinstead J (1995) "Risk Assessment and Contained Use of Genetically Modified Microorganisms", in Genetically Modified Organisms: A Guide to Biosafety, Tzotzos GT (Ed) CAB International, Oxford, ISBN 0 85198 972 1, p28

6. modified organisms are generally used under conditions of 'Good Industrial Large Scale Practice' (GILSP) defined by a working group of OECD.

7. Advisory Committee on Genetic Modification: Laboratory Containment Facilities for Genetic Manipulation ACGM/HSE Note 7 (1993) UK. These notes are currently being re-written to form a compendium of advice on the contained use of modified organisms, and should be available in the new form during 1997.

8. Advisory Committee on Genetic Modification: Laboratory Containment Facilities for Genetic Manipulation ACGM/HSE Note 8 (June 1988) UK.

9. Advisory Committee on Genetic Modification: Laboratory Containment Facilities for Genetic Manipulation ACGM/HSE Note 8 (June 1988) UK.

10. Organisation of Economic Co-operation and Development Group of National Experts on Safety in Biotechnology. 1986, ISBN 92-64-12857-3.

11. Advisory Committee on Genetic Modification: Laboratory Containment Facilities for Genetic Manipulation ACGM/HSE Note 6 (1987) UK.

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