Deliberate Release Research Articles

GMO Monitoring System in Lithuania

The development of a National GMO Monitoring System largely depends upon the provisions of the Order on Regulation for Preparation of General Surveillance and Monitoring Plan of GMOs and/or GMPs after placing on the market (postmarket monitoring) (adopted on 01/12/2003, No.601). GMOs and GMPs user is responsible for the gathering of monitoring data, analysis and their quality assurance during the experimental phase of deliberate release into the environment, before placing on the market. Reports on monitoring programmes and data obtained are communicated to the competent Authorities (CA) of other member States (MS) and to the EC through the GMO Information system, administered by the Ministry of Environment. The State Food and Veterinary Service is responsible for the control of monitoring. The National Veterinary Laboratory has started to implement a monitoring programme for the state control of labelled and unlabelled food products for the content of GMOs since 2004, the volunteer GM canola control – since 2007.

The German ZKBS providing advice for risk mamagers since 1978

The German Central Commission on Biological Safety (ZKBS) is a body of honorary experts, examining genetically modified organisms (GMOs) for possible risks to humans and the environment and is responsible for issuing statements to this end. Statements of the ZKBS constitute recommendations that should be taken into consideration. Any deviation from the recommendations of the ZKBS requires justifying explanations. The office of the ZKBS is in the BVL (Federal Office of Comsumer Protection and Food Safety). Its duties comprise the assessments of the safety of genetic engineering operations and facilities as well as the assessments of the safety of deliberate release into the environment including placing on the market of GMO. With the amendment of theGenetic EngineeringAct inApril 2008, thenumber of experts was raised to twenty members and twenty deputy members representing different scientific fields and public interests. The commission adopts its resolutions either in written proceedings or in closed sessions that are held approximately once in every two months at the BVL, to which the involved authorities and the Federal Ministries are invited. A report of activities is published each year, for the purpose of informing the public.

Experimental acute respiratory Burkholderia pseudomallei infection in BALB/c mice

Burkholderia pseudomallei is the causative agent of melioidosis, which is considered a potential deliberate release agent. The objective of this study was to establish and characterise a relevant, acute respiratory Burkholderia pseudomallei infection in BALB/c mice. Mice were infected with 100 B. pseudomallei strain BRI bacteria by the aerosol route (approximately 20 median lethal doses). Bacterial counts within lung, liver, spleen, brain, kidney and blood over 5 days were determined and histopathological and immunocytochemical profiles were assessed. Bacterial numbers in the lungs reached approximately 10(8) cfu/ml at day 5 post-infection. Bacterial numbers in other tissues were lower, reaching between 10(3) and 10(5) cfu/ml at day 4. Blood counts remained relatively constant at approximately 1.0 x 10(2) cfu/ml. Foci of acute inflammation and necrosis were seen within lungs, liver and spleen. These results suggest that the BALB/c mouse is highly susceptible to B. pseudomallei by the aerosol route and represents a relevant model system of acute human melioidosis.

The status of agricultural biotechnology and biosafety in Romania.

Since 2000, Romania had in place a legal Biosafety Framework with the objective of protecting human health and the environment, in accordance with the precautionary principle (Government Ordinance 49/2000). In 2000, Romania was the only country in Europe approving market release of two genetically modified (GM) crops: Roundup Ready (RR) soybean and Superior New Leaf potato. GM potato has not been commercialized. On the contrary, RR soybean was grown on thousands of hectares. Romania is one of the few European countries with favourable conditions for soybean production and, in 2006, was one of the nine countries in the world that cultivated this GM crop. As member of the European Union beginning with 2007, Romania must comply with the rules for placing on the market, traceability and labelling of genetically modified organisms (GMOs) as laid down by EU legislation. Consequently, as of 2007, Roundup Ready soybean cultivation was banned in Romania. Today, Bt-maize expressing the insecticidal protein Cry1Ab from Bacillus thuringiensis (Bt) in the only GM crop approved for cultivation in the EU. In 2007, Bt hybrids were grown on about 300 ha and in 2009, on about 3000 ha, especially in areas where the lepidopterean target pest caused serious infestations. In this overview, we address the current status of Romanian biosafety legislation, agricultural biotechnology research, deliberate release and commercialization of GM crops. We also present the main stakeholders and their expectations regarding the development of agricultural biotechnology in Romania

The usefulness of total concentrations and pore water concentrations of pesticides in soil as metrics for the assessment of ecotoxicological effects ‐ Scientific Opinion of the Panel on Plant Protection Products and their Residues (PPR)

The usefulness of total concentrations and pore water concentrations of pesticides in soil as metrics for the assessment of ecotoxicological effects ‐ Scientific Opinion of the Panel on Plant Protection Products and their Residues (PPR)

Postexposure Immunization with Modified Vaccinia Virus Ankara or Conventional Lister Vaccine Provides Solid Protection in a Murine Model of Human Smallpox

Decades after the cessation of smallpox vaccination, the potential of the deliberate release of pathogenic orthopoxviruses has forced a reconsideration of using these extremely efficient human vaccines. Scenarios of sudden biothreats have prompted demand for rapidly protective vaccination. However, the feasibility of short-term vaccination (i.e., vaccination shortly before exposure) with vaccinia virus (VACV) is uncertain. We tested the rapid protective capacity of vaccines based on VACV strain Lister (VACV-Lister) and on modified VACV Ankara (MVA) in different mouse models, comparing lethal infections with VACV strain Western Reserve (VACV-WR) or ectromelia virus (ECTV). In contrast to VACV-WR challenge, we found extended incubation periods after ECTV challenge, allowing successful therapeutic immunization with VACV-Lister and MVA when applied 2-3 days after exposure. Rapid protection from respiratory tract ECTV infection was significantly affected by vaccine dose and was associated with occurrence of poxvirus-specific antibodies. Vaccinations in type I interferon receptor-deficient mice were protective, whereas recombination activating gene 1-deficient mice lacking mature T and B cells failed to mount immunity after short-term vaccination, confirming an essential role of adaptive immune responses. ECTV infection in mice models the course of human smallpox. Our data provide evidence to substantiate historical data on the usefulness of postexposure vaccination with conventional VACV and the new candidate MVA to protect against fatal orthopoxvirus infections.

Stable implantation of orthogonal sensor circuits in Gram‐negative bacteria for environmental release

A broad host range, orthogonal genetic platform has been developed to format sensor circuits in the chromosome of Gram-negative microorganisms destined for environmental release as bioindicators of toxic or perilous compounds (e.g. explosives) in soil. The genetic scheme includes the generation of a genomic landing pad for the sensor module with a Tn5-mini-transposon bearing an optimal attTn7 sequence and a choice of reporter systems with optical and enzymatic outputs. The array of functional elements thereby inserted in the chromosome match that of a cognate plasmid vector which delivers the transcription factors and the promoters to a frame that places the regulatory parts in front of the reporters. Site-specific recombination sites allow the deletion of antibiotic resistances and enables reporter output prior to deliberate release. The system thus allows the production and maintenance of cells in a pre-release state and its intentional conversion in deliverable strains that fulfil all safety, stability and performance criteria. The combination of such a genetic platform with a variant of the transcriptional regulator XylR of Pseudomonas putida that responds to 2,4-dinitrotoluene has been the basis for the production of strains that emit light upon exposure to residues of explosives in a soil microcosm.

The threat to Emergency Departments from bioterrorism in Australia

The Commonwealth Department of Health (DoHA) has recently developed two lists of threat agents in consultation with relevant stakeholders around Australia. These have been labeled Tier 1 and Tier 2 agents. This list includes bacteria, viruses, and toxins derived from bacteria and plants. Legislation will soon be introduced to introduce controls on laboratories who store these agents. This presentation will look at the agents on these two lists and examine those that represent the biggest threat in the Australian context, detailing their clinical presentation, their mode of spread and how they might be used as bioterrorist agents. A number of these agents produce flu-like symptoms initially, making diagnosis difficult. This has important ramifications for patients, who will die unless antibiotic therapy is initiated rapidly. The discussion will concentrate on the threat theses agents pose to emergency staff when receipting patients exposed to a deliberate release of agent. An attack of this nature would quickly overwhelm even large Emergency Departments, not only from presentations of seriously ill patients but also from the large numbers of worried well who will present. Whether the agent is infectious or not will have serious ramifications for ongoing spread of the agent throughout the community.

Mathematically Assessing the Consequences of Food Terrorism Scenarios

We derive mathematical expressions for the mean number of casualties resulting from a deliberate release of a biological or chemical agent into a food supply chain. Our analysis first computes the amount of contaminated food as a function of the network topology and vessel sizes in the food processing plant. A probabilistic analysis, in which each potential consumer of contaminated food has his own random purchase time, infectious dose, and incubation period, determines the number of people who consume enough tainted food to get infected or poisoned before the attack is detected and food consumption is halted. These simple formulas can be used by the U.S. government and the food industry to develop a rough-cut prioritization of the threats from food terrorism, which would be a 1st step toward the allocation of appropriate prevention and mitigation resources.

Nature Protection and the Introduction into the Environment of Genetically Modified Organisms: Risk Analysis in EC Multilevel Governance

Genetically modified organisms (GMOs) are usually introduced into the environment through experimental releases or extensive cultivation. The risks involved are controlled via both the authorization for deliberate release and the authorization for the placing on the market of a GMO. In these two steps, the core issues of legal protection are public health and the environment. GMO-free agriculture has been introduced as a third – economic – protected good. This has triggered coexistence measures keeping GM and non-GM agriculture separated. Given the fact that much has already been said about risks of GMOs to human health, this article will rather focus on the environmental dimension, and more precisely on aspects of nature protection. It will accordingly explore whether nature is adequately protected through the authorization for the release and the authorization for the market placement of GMOs. While these questions concern regimes aiming at preventing environmental damage, co-existence, if related to natural ecosystems (rather than to the protection of a variety of agriculture), raises the question of whether ecosystems can be considered as protectable goods even if no traditional damage is caused.

Zoonoses Likely to Be Used in Bioterrorism

Bioterrorism is the deliberate release of viruses, bacteria, or other agents used "to cause illness or death in people, animals, or plants. Only modest microbiologic skills are needed to produce and effectively use biologic weapons. And biological warfare has afflicted campaigns throughout military history, at times playing an important role in determining their outcomes. There is a long list of potential pathogens for use by terrorists, but only a few are easy to prepare and disperse. Of the infectious diseases, the vast majority are zoonoses. The Centers for Disease Control and Prevention's highest-priority bioterrorism agents are in Category A. The only disease that does not affect animals in Category A is smallpox, which was eliminated by a worldwide vaccination program in the late 1970s. Because these diseases can infect animals and humans, the medical and veterinary communities should work closely together in clinical, public health, and research settings.

Botulismus

In times of increased terrorist threat health professionals need to be prepared for bioterrorist events. The goal must be to give doctors an overview over the current state of knowledge and risk assessment of botulinum toxin. This review is based on Information gathered by a systematic analysis of the literature and by contacting experts. The toxicity of botulinum toxin exceeds any other known natural toxin. Clinical features of botulism consist of an acute, afebrile symmetrical and descending paralysis, regardless of the route of exposure, with normal mental status, sensory functions and electrolyte values. The initial diagnosis is often wrong in individual cases of botulism, but clusters with typical symptoms and two or more cases usually provide the diagnosis. Current treatment is primarily supportive care, respiratory support and antitoxin administration. Early application of antitoxin can limit the extent of the paralysis, but will not reverse it. Antitoxin for adult patients is of equine origin, while children in the USA can be treated with a recently developed human antitoxin. A pentavalent toxoid vaccine is available for persons at high risk of exposure. Botulinum toxin is easily extracted and ubiquitously available. These two features, together with the high toxicity, makes misuse easy. Misuse will continue to occur. Although a rare disease in Western Europe, botulism should be included in the differential diagnosis in patients with specific symptoms of paralysis. There is the potential threat of deliberate release of botulinum toxin. For this reason every outbreak of botulism must be assessed for any possible links to terrorism.

Grasping at the routes of biological invasions: a framework for integrating pathways into policy

SummaryPathways describe the processes that result in the introduction of alien species from one location to another. A framework is proposed to facilitate the comparative analysis of invasion pathways by a wide range of taxa in both terrestrial and aquatic ecosystems. Comparisons with a range of data helped identify existing gaps in current knowledge of pathways and highlight the limitations of existing legislation to manage introductions of alien species. The scheme aims for universality but uses the European Union as a case study for the regulatory perspectives.Alien species may arrive and enter a new region through three broad mechanisms: importation of a commodity, arrival of a transport vector, and/or natural spread from a neighbouring region where the species is itself alien. These three mechanisms result in six principal pathways: release, escape, contaminant, stowaway, corridor and unaided.Alien species transported as commodities may be introduced as a deliberate release or as an escape from captivity. Many species are not intentionally transported but arrive as a contaminant of a commodity, for example pathogens and pests. Stowaways are directly associated with human transport but arrive independently of a specific commodity, for example organisms transported in ballast water, cargo and airfreight. The corridor pathway highlights the role transport infrastructures play in the introduction of alien species. The unaided pathway describes situations where natural spread results in alien species arriving into a new region from a donor region where it is also alien.Vertebrate pathways tend to be characterized as deliberate releases, invertebrates as contaminants and plants as escapes. Pathogenic micro‐organisms and fungi are generally introduced as contaminants of their hosts. The corridor and unaided pathways are often ignored in pathway assessments but warrant further detailed consideration.Synthesis and applications.Intentional releases and escapes should be straightforward to monitor and regulate but, in practice, developing legislation has proved difficult. New introductions continue to occur through contaminant, stowaway, corridor and unaided pathways. These pathways represent special challenges for management and legislation. The present framework should enable these trends to be monitored more clearly and hopefully lead to the development of appropriate regulations or codes of practice to stem the number of future introductions.

Notification (Reference C/NL/06/01) for the placing on the market of the genetically modified carnation Moonaqua 123.8.12 with a modified colour, for import of cut flowers for ornamental use, under Part C of Directive 2001/18/EC from Florigene ‐ Scientific Opinion of the Panel on Genetically Modified Organisms

Notification (Reference C/NL/06/01) for the placing on the market of the genetically modified carnation Moonaqua 123.8.12 with a modified colour, for import of cut flowers for ornamental use, under Part C of Directive 2001/18/EC from Florigene ‐ Scientific Opinion of the Panel on Genetically Modified Organisms

Gene and Cell Therapy in Germany and the EU

Gene and somatic cell therapy medicinal products as well as tissue engineered products are summarized under the term advanced therapy medicinal products in the EU. The Paul-Ehrlich-Institut has established a specialized division with various sections providing scientific advice and clinical trial authorization to applicants, thereby protecting patients’ health and supporting product development. Gene therapy medicinal products include products suitable for in vivo diagnostics, for the prevention of infectious diseases (such as vaccines) and the treatment of common diseases such as cancer, cardiovascular and infectious diseases, and of rare or orphan diseases such as inherited monogeneic diseases. Clinical trial authorization in Germany is provided by the Paul-Ehrlich-Institut and, if genetically modified organism-containing medicinal products are used, includes deliberate release authorization under consultation with the Federal Office for Consumer Protection and Food Safety (BVL). Firstmarketing authorization applications of gene therapy medicinal products developed for the treatment of cancer have been submitted to the European Medicines Agency (EMEA) and have been or are being reviewed by experts from national medicines agencies. Beneficial gene therapy treatment in clinical trials has been reported for infants suffering from adenosine deaminase deficiency-based severe combined immunodeficiency disease. Human somatic cell therapy medicinal products are mainly being developed for cancer immunotherapy; tissue engineered products contain viable cells able to replace, repair or substitute for human tissues or cells, e.g., after myocardial infarction. Chondrocytes are being used clinically for cartilage repair and an application for EU-wide marketing has been submitted to the EMEA. The main issues for cell-containing medicinal products are cell selection, differentiation, manipulation to obtain specific characteristics and avoiding pre-neoplastic alterations. In summary, special expertise is needed to develop advanced therapy medicinal products and has to be met by scientists specialized in regulatory aspects to support patient safety and product development. Consequently, the EMEA will establish the Committee for Advanced Therapies to review future marketing authorization applications.

Recent progress on environmental biosafety assessment of genetically modified trees and floricultural plants in Japan

A broad spectrum of living modified organisms (LMOs) have been developed and commercial production of them have already been started. It is the global requirement to conduct environmental biosafety after the implementation of Cartagena Protocol on Biosafety. Environmental biosafety assessment is of cardinal importance even in confined field trials and at the deliberate release to an environment for practical uses. It is necessary to collect various categories of data prior to fields; not only about the morphological and physiological traits of the LMOs in some confined experimental facilities, but also about characteristics of host organisms in the natural habitat and field for intended releases. Environmental release of LMOs is possible only after collected information is examined systematically and comprehensively in a scientifically-sound manner, and target end-points of such uses are well-identified. Environmental biosafety assessments principally require case-by-case evaluation. Gene-flow, allelopathy and invasiveness are three key aspects in GM plants. In Japan, many crop species do not have their wild relatives due to their historical introduction from overseas. Furthermore, crops grow properly only under agricultural practices, and invasive weediness of these plants is very likely low. In contrast, floricultural plants and trees have related native species located around the fields that can cross with the transgenic counterparts in many cases. In this review, we examine the environmental biosafety assessment of LMO floricultural plants and trees enumerating the example of field trials in Japan, and describe concepts that should be noted for the commercial cultivation and environmental release of these species.

Take-home messages

• Post Market Environmental Monitoring (PMEM) is mandatory in all applications for deliberate release submitted under Directive 2001/18/EC and EU Regulation 1829/2003. • PMEM aims at identifying possible unanticipated adverse effects on human health or the environment which could arise directly or indirectly from GM plants. • PMEM is composed of case-specific monitoring (CSM) and general surveillance (GS). CSM is notmandatory butmaybe required to verify the environmental risk assessment while GS must be part of an application. • EFSA is responsible for assessing the scientific quality of PMEM plans submitted with each application. • EFSA has adopted guidance to applicants for developing PMEM plans (adopted December 2005). • The EFSA GMO Panel is of the opinion that GS cannot be driven by specific hypotheses (cause-effect) and should, when possible, make use of existing monitoring programmes in addition tomore focussedmonitoring systems as farm questionnaires. • Data quality, management and statistical analysis are of high importance in the design of a GS plan and comparison should be made with baseline data. • EFSA recommends early contact between applicants and Member States (selection of existing networks) and recommends harmonisation and coordination of data collection and analysis. • PMEM is a risk management measure. • The Commission and the Member States via their Competent Authorities are responsible for the PMEM and take the decisions. • The body in charge of proposing harmonisation of PMEM is the EU Monitoring Working Group. • A pragmatic PMEM for MON 810 that addresses the proposedprotectiongoals has been implemented successfully. • The 2005 monitoring report for MON 810 confirms the conclusion of safety reached in the environmental risk assessment. • The future presence of multiple events and stacks on the market will require: – some adaptation, including modularity, – a harmonised approach across the industry that has started under the EuropaBio banner.

First EFSA experiences with monitoring plans

A plan for Post Market Environmental Monitoring (PMEM) of genetically modified (GM) plants is mandatory in all applications for deliberate release submitted under EU Directive 2001/18/EC and EU Regulation 1829/2003. PMEM is composed of case-specific monitoring and general surveillance of GM plants. The European Food Safety Authority (EFSA) is responsible for assessing the scientific quality of PMEM plans submitted with each application. In a scientific opinion, the EFSA GMO Panel presented specific guidance for applicants for developing PMEM plans. In addition the EFSA GMO Panel explained the scientific rationale for this guidance and makes a number of recommendations for the management and conduct of PMEM by both applicants and risk managers. Until the end of March 2007, the EFSA GMO Panel gave opinions on 11 PMEM plans submitted under EU Directive 2001/18/EC and another 11 plans submitted within applications under EU Regulation 1829/2003. Currently 27 applications for GM plants are still in the evaluation process and the EFSA GMO Panel sent 26 questions to the applicants for clarification and additional information during the evaluation process. Sixteen of these questions were related to General Surveillance (e.g. general structure, farm & operator questionnaires, integrations of identity preservation systems, use of existing surveillance systems, feral plant surveillance). Nine questions requested clarification on the environmental risk assessment, which might effect whether case-specific monitoring is needed. In one case (an application for cultivation of a GM crop), the GMO Panel recommended case-specific monitoring. We explain in more detail how the environmental risk assessment of two Bt maize dossiers (maize Bt11 and 1507) were considered by the GMO Panel in the evaluation of the PMEM plan and the consequences for the environmental monitoring plan. As stated in the EFSA opinion on post-market environmental monitoring, the general surveillance plans shall, when possible, make use of existing monitoring systems in addition to more focused monitoring systems. However, the use of these national monitoring programmes is outside of the management and control of an individual applicant and thus it cannot be the task of an applicant alone to use, modify or improve existing surveillance systems. The availability of biodiversity monitoring programmes in the EU Member States should be evaluated by applicants in close liaison with risk managers.

Gene therapy legislation in The Netherlands

Several regulatory organisations are involved in the assessment of clinical gene therapy trials involving genetically modified organisms (GMOs) in The Netherlands. Medical, ethical and scientific aspects are, for instance, evaluated by the Central Committee on Research Involving Human Subjects (CCMO). The Ministry of Housing, Spatial Planning and the Environment (VROM) is the competent authority for the environmental risk assessment according to the deliberate release Directive 2001/18/EC. A Gene Therapy Office has been established in order to streamline the different national review processes and to enable the official procedures to be completed as quickly as possible. Although the Gene Therapy Office improved the application process at the national level, there is a difference of opinion between the EU member states with respect to the EU Directive according to which gene therapy trials are assessed, that urges for harmonisation. This review summarises the gene therapy legislation in The Netherlands and in particular The Netherlands rationale to follow Directive 2001/18/EC for the environmental risk assessment.

Development of a yeast biosensor–biocatalyst for the detection and biodegradation of the organophosphate paraoxon

Organophosphate (OP) poisoning can occur through unintentional exposure to OP pesticides, or by the deliberate release of OP nerve agents. Consequently, there is considerable interest in the development of systems that can detect and/or biodegrade these agents. The aim of this study was to generate a prototype fluorescent reporter yeast biosensor that could detect and biodegrade the model OP pesticide, paraoxon, and subsequently detect paraoxon hydrolysis. Saccharomyces cerevisiae was engineered to hydrolyze paraoxon through the heterologous expression of the Flavobacterium species opd (organophosphate degrading) gene. Global transcription profiling was subsequently used to identify yeast genes, which were induced in the presence of paraoxon, and genes, which were associated with paraoxon hydrolysis. Paraoxon-inducible genes and genes associated with paraoxon hydrolysis were identified. Candidate paraoxon-inducible promoters were cloned and fused to the yeast-enhanced green fluorescent protein (yEGFP), and candidate promoters associated with paraoxon hydrolysis were fused to the red fluorescent protein (yDsRed). The ability of the yeast biosensor to detect paraoxon and paraoxon hydrolysis was demonstrated by the specific induction of the fluorescent reporter (yEGFP and yDsRed, respectively). Biosensors responded to paraoxon in a dose- and time-dependent manner, and detection was rapid (15 to 30 min). yDsRed induction occurred only in the recombinant opd(+) strains suggesting that yDsRed induction was strictly associated with paraoxon hydrolysis. Together, these results indicate that the yeast biocatalyst-biosensor can detect and degrade paraoxon and potentially also monitor the decontamination process.