EN 12685-1998 en Biotechnology - Modified Organisms for Application in the Environment - Guidance for the Monitoring Strategies for Deliberate Releases of Genetically Modified Micr.pdf

1、 STD.BSI BS EN LZbAS-ENGL 1998 Lb24bb9 07395b7 b28 BRITISH STANDARD Biotechnology - Modified organisms for application in the environment - Guidance for the monitoring strategies for deliberate release of genetically modified micro-organisms, including viruses The European Stanard EN 12685:19!38 has

2、 the status of a British Standard ICs 07.080; 07.100.01 BS EN 12685: 1998 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGH LAW STD-BSI BS EN 12b85-ENGL 1778 LbZLibbS 07375b8 5bq been prepared under the direction of the Sector AmdNo. Committee for Materials and Chemicals, was publish

3、ed under the authority of the Standards Committee and comes into effect on 16 December 1998 Q BSI 1998 ISBN O 880 30178 8 BS EN 12683:1998 Date Textaffected National foreword This British Standard is the English language version of EN 12685:1998. The UK participation in its preparation was entnisted

4、 to Technical Committee CW, Biotechnology, which has the responsibility to: - aid enquirers to understand the text; - present to the responsible European committee any enquiries on the - monitor related internationai and European developments and promulgate interpretation, or proposals for change, a

5、nd keep the UK interests informed; them in the UK. A iist of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement internationai or European publications referred to in this document may be found in the BSI Sta

6、ndards Catalogue under the section entitied “international Standards Correspondence index“, or by using the “Find“ facility of the BSI Standards Electxonic Catalogue. A British Standard does not purport to include ail the necessary provisions of a contract Users of British Standars are responsible f

7、or their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN titie page, pages 2 to 10, an inside back cover and a back cover. STD-BSI BS EN 12bA5-ENLL

8、 1778 m lbZibb7 07375b7 LiTO m EUROPEAN STANDARD EN 12685 NO= EUR0PEE”E EXJROP 07.100.01 Descriptors: biotechnolo, genetics, modified organisms, environments, environmental protection, inspection, experimental design English version Biotechnology- Modied organism for application in the environment -

9、 Guidance for the monitorin$ strategies for delibemte releases of genetically modied rnicmganisms, including viruses Biotechnologie - Organismes modins dissmin - the monitoring of GMMs for food, human health and veterinary applications. NOTE Attention is drawn to national, European and international

10、 regulations, and relevant standards covering the monitorinp of GMMs in food, human health and veterinary applications. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriat

11、e places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this European Standard oniy when incorporated in it by amendment or revision. For undated references the latest edition of the publication

12、 referred to applies. Page 3 EN 12685:1998 EN 12686, Biotechnology - Modified organ.isms for application in ULe environment - Guzdance fw the sampling stmtegies for cid%erate releases of genetically modified micro-organisms, including viruses. 3 Denitions For the purposes of this standard, the follo

13、wing definitions apply: 3.1 analyte substance sought or determined 3.2 behaviour interaction of the organism(s) with abiotic and biotic environments, its (their) occurrence, persistence, multiplication and spreading abilities 3.3 control preparation of known characteristics used to standardize an an

14、alysis 3.4 detection recognition of the presence of an organism or of a moleculas structure wih a sample 3.6 genetic modification of interest conceptual design for altering the genetic material within an organism NOTE 1 The genetic modification of interest can be described at different levels of mol

15、ecular detail. NOTE 2 The conceptual design can include insertion, substitution or deletion of genetic material. 3.6 genetically modified micro-organism micro-organism in which the genetic b) validalion of the monitoring protocol; c) execution of the monitoring protocol; d) record keeping. Responsib

16、ility for the steps should be assigned to a specic authoriu, organization or person. The monitoring strategy should be reviewed reguiarh in the light of the inspections of the release site, to ensure its continuing validity 6.2 Criteria for the design of the monitoring protocol The following key fac

17、tom should be considered in the initial design of the protocol to ensure the design correlates with the monitoring strategy objectives as determined by the person designing the test a) sampling strategy appropriate to the objectives and needs of the monitoring strategy as described in EN 12686; NOTE

18、 The development of a monitoring and sampling valid strategv for deliberate releases of genetically modified micro-organisms in the environment is summarized in Figure A. 1. b) extent of monitoring necessary to fuli the requirements of the experiment such as timecale, scale and area of sampling; c)

19、predicted behaviour of GMM, pasticulariy with respect to competitive abiiw, dispersal, persistence and the ability to fonn spores, resting forms and other specialized siructures; d) relevant area for monitoring in line with the monitoring Objectives such as the release site andor the potential dispe

20、rsal area; e) particular features of the release site to monitor such as adjacent Sites, stseams, soil movement activities, meteorological parameteq soil properties; f) presence of potential hosts, presence of potential microbial vectors; g) appropriate choice of monitoring methods. O BSI 1998 STD.B

21、S1 BS EN LZb5-ENGL 1778 Ri LbZqbb 0737573 7ZI, m Page 5 EN 12685:1998 6.3 Validity of the design of the monitoring protocol 6.3.1 General Factors and criteria which should be considered for the determination of the suitabiity and validity of a monitoring protocol are given in 6.3.2 to 6.3.6. The fol

22、lowing lists of factors and criteria should not be considered as exhaustive. 53.2 Considerations with respect tofiWl1ling the overall experimental objective in fulfilment of the overall experimental objective, the aim of individual experiments can be: - detection; - identifidon; - determination of t

23、he molecular stability of the gene expressioq - determination of the effect of the modificatioq - rapid screening or detailed investigation; - quantitative or qualitative approach. 6.3.3 considerations with respect to the genetic modification of interest The factor) being monitored can be one or a c

24、ombinatjon of the following: - gene presence; - gene expression; - presence of the GMM; - behaviour of the GMM in the environment. 63.4 Considerations with respect to the release site Factors influencing the monitoring strategy with respect to the release site can be: - methods of application of the

25、 GMM to the release site; - environmental factors such as season, weather; - biological factors such as predation or the presence of organisms which could displace the GMM, hosts, or alternatively serve as microbial VeCtors; - physical factors such as soil type, water drainage; - presence of populat

26、ions related to the GMM. 6.3.6 Considerations with respect to the validity of monitoring methods 6.3.6.1 KI/ considemtwns wit% respect to .?%e validity of monitoring methods The following method validation parameters should be considered to ensure that the performance charader - feasibiliw, - limita

27、tions; - access to appropriate controls; - selectivity and specificity; - reproducibiliQq - repeatability; - limit of detection; - reliability. NOTE A full definition of several of these parameters is given in Eurachem guidance document no 1 - 1993 (see Annex B 3). The following explanations are int

28、ended as a guide for the application of this European Standard. a) Fit for purpose should address the specific experimental needs and is relevant to the aim, scope and available resources of the experiment. b) Feasibility indicates how workable the method is in practice. c) Limitations are the short

29、comings of a method and any restrictions to use. d) Access to appropriate controls should include the parent or the non-modified recipient organism. e) Selectivity of a method refers to the extent to which it can determine particulas dyte(s) in a complex mixture without interference from the other c

30、omponents in the mixture. Specificity qualifies a method which is selective for an analyte or group of anal- of interest. f) Reproducibility is the closeness of the agreement between the results of successive measurements carried out independently using an identical experimental protocol but under c

31、hanged conditions. The changed conditions can include operator, location and time. g) Repeatability is the closeness of the agreement between the resuits of successive measurements carried out under the same conditions. Repeatability conditions include the same operator, the same location and repeti

32、tion over a short period of time. h) Limit of detection is the smallest amount of analyte that can be reliably detected This issue implies the concept of sensitivity. i) Reliability qualifies a dependable method which gives both repeatable and reproducible results. 6.3.6.2 Further factors influencin

33、g the choice of did monitoring mthods The following considerations can also be relevant to the choice of valid methods used for monitoring, depending on the nature and objectives of the monitoring experiment. Not ail the considerations are applicable for each experimental situation. a) Many methods

34、can be used to monitor GMMs released into the field They can include microbiological, virological andor molecular biological methods. For any method, an appropriate sampling regime and, if nece-, transport arrangements should be adopted (see EN 12686). b) The methods used for monitoring required can

35、 vary from release depending primarily upon the monitoring objectives and should be identified in accordance with the validity criteria identified in 6.3. O BSI 1998 Page 6 EN 12685:1998 c) The choice of methods used for monitoring depends upon the purpose for which the monitoring is done and should

36、 enable detection of events under study. The nature of the GMM and its hosts can help to determine the methods used for monitoring a release; iferent types of GMM can require different techniques for monitoring. Key factors to be considered in the establishment of a monitoring strategy would include

37、: - environmental location, e.g. air, soil, water; - type of GMM, e.g. bacteria, fungi; - type and number of hosts; - state of GMM, e.g. spore, vegetative or “dormant” cells; - tmnsmjssion of GMM e.g. seedborne, aphid tsansmission; - any symbiotic relationship; - environmental persistence. Thus GMMs

38、 dispersed through the air can require very Werent sampling and detection techniques from soil micro-organisms or those with a symbiotic relationship with another, higher organism. d) The validity of any one method, or combination of methods, can be affected by the ease and accuracy of identificatio

39、n of the introduced GMMs andor the introduced genes. Identification of GMMs should iddy be by means of easily recognizable phenotypic or genotypic characteristics (see Annex B e) lb facilitate monitoring, the released GMM should be unequivocally recognizable. Any idenmg C-C should ideally be stable,

40、 and clearly different from the equivaent characteristic displayed by local microbial communities or virai popuations. Marker characteristics that are easy to identify can be suitable for assessing the spread of the GMM or horizontal transfer of genetic material. f) For some releases it cannot be po

41、ssible to detect GMMs by direct recovery Once released into the environment some microorganisms can undergo physiological changes which lead to them bei no longer culturable under standard laboratory conditions. Alternative monitoring techniques can be at the DNA-level, in which case the experiment

42、can need to demonstrate that the DNMA target is associated with or derived from GMMs contained in the sample, or be related to the effect) the GMM should have, for example its effects as a biocontrol agent. g) In many cases, monitoring can need an estimation of the population size of the released GM

43、Ms. Suitable, quantitative methods can therefore be nec-, and appropriate statistical anaysii methods can also be required Such data, gathered over time can indicate whether the released population is survMng, multiplying or declining. 41, 51). h) Factors to be taken into account include the ease an

44、d simplicity of conventional cultural methods for microbiology and virology and their ability to screen iarge numbers of samples, but sensitivity and application to viable nonahurable micro-organisms could be iimited Molecular ampijfcation techniques are usually more rapid and sensitive, but cannot

45、readily be used to estimate population sizes and are very prone to inhibition and reduced detection limits due to typical environmentai malxix components. Moreover, they do not generally give an inidon of the potential viability of the GMM. i) Although observation of the trial site is unlikely to be

46、 able to provide confirmation of the presence of the released GMM, this observation can give the ht indication of an expected or unexpected effect. j) The transfer of the inserted gene(s) from the released GMM to a resident recipient should result in the appeamnce of the gene) of interest in a new g

47、enetic background. For example, for vinis-, the inserted gene can code for an insect specific toxin, insect hormone, pharmaceutidy active protein; for bacteria and fungi, the inserted gene can code for heavy metal or other resistance, or the ability to degrade a particular xenobiotic compound Micro-

48、organisms possessing the genes can be able to grow on the appropriate selective or elective media containing the particular heavy metal, antibiotic or xenobiotic compound if the released GMM has a well-defined genetic background such as chromosomal markers, the appearance of characteristics of the t

49、ransgene(s) in a different genetic background can indicate that the transgenes have been transferred into (resident) recipient organisms. If the released GMM does not have a well-defined genetic background, the appearance of the -gene in a recipient organism should be verified by appropriate molecular methods. However, it should be noted that in environmental samples such as soil, the majority of viable micro-organisms present cannot be cultured by conventional laboratory techniques, or cannot expres the transferred gene. In such cases, appropriate molecular m