chapter2.6

英文论文提纲的格式范文chapter i introduction 11.1 research background 11.2 significance and objectives of the research 21.2.1 the significance of the research 21.2.2 the objectives of the research 21.2.3 summary 31.3 studies of fossilization both home and abroad 31.3.1 studies of fossilization abroad 41.3.2 studies of fossilization in china 51.3.3 summary 61.4 organization of the thesis 7chapter ii literature review 72.1 interlanguage 82.2 the definitions of fossilization 92.3 the classification of fossilization : :. 132.3.1 individual fossilization vs. group fossilization 132.3.2 temporary fossilization vs. permanent fossilization 14 2.3.3 how to identify fossilization 152.4 possible factors of fossilization 172.4.1 internal factors 182.4.2 external factors 202.5 error analysis 222.5.1 the background of error analysis 222.5.2 procedures of error analysis 232.5.3 classification of errors 242.5.4 the significance of error analysis 262.6 summary 26chapter iii design of the research 273.1 research questions 273.2 subjects 283.3 method 283.4 instruments 293.5 procedures 293.6 summary 30chapter iv result and discussion 314.1 data analysis 314.1.1 classification of errors 314.1.2 analysis of the errors 314.2 the design of the questionnaires 374.3 the analysis of the questionnaires 384.4 analysis of causes of fossilized errors in writing 40 4.5 summary 42chapter v strategies for fossilization avoidance 435.1 to take a good attitude to the interference of li 43 5.2 to enhance the learners' motivationr445.3 to guarantee the quality and quantity of l2 input 46 5.4 to provide effective feedback 485.5 to attach more importance to grammar teaching 49 5.6 to apply more effective learning strategies 495.7 summary 50chapter vi conclusion 516.1 a brief review of the study 516.2 major findings 526.3 limitations of this study 536.4 suggestions 54bibliography 55。

国际危规IMDG code (Amdt.35-10)Foreword...Preamble…Part 1 – general provisions, definitions and trainingChapter 1.1 general provisions1.1.0 introductory notes1.1.1 application and implementation of the code1.1.2 conventions1.1.3 dangerous goods forbidden from transportChapter 1.2 definitions, units of measurement and abbreviations1.2.1 definitions1.2.2 units of measurement1.2.3 list of abbreviationsChapter 1.3 training1.3.0 introductory note1.3.1 training of shore-side personnelChapter 1.4 security provisions1.4.0 introductory note1.4.1 general provisions for companies, ships and port facilities1.4.2 general provision for shore-side personnel1.4.3 provisions for high consequence dangerous goodsChapter 1.5 general provisions concerning class 71.5.1 scope and application1.5.2 radiation protection program1.5.3 quality assurance1.5.4 special arrangement1.5.5 radioactive material possessing other dangerous properties1.5.6 non-compliancePart 2 – classificationChapter 2.0 introduction2.0.0 responsibilities2.0.1 classes, divisions, packing groups2.0.2 UN numbers and proper shipping names2.0.3 classification of substances, mixtures and solutions with multiple hazards (precedence of hazard characteristics)2.0.4 transport of samplesChapter 2.1 class 1 – explosives2.1.0 introductory notes2.1.1 definition and general provisions2.1.2 compatibility groups and classification codes2.1.3 classification procedureChapter 2.2 class 2 – gases2.2.0 introductory note2.2.1 definitions and general provisions2.2.2 class subdivisions2.2.3 mixtures of gasesChapter 2.3 class 3 – flammable liquids2.3.0 introductory note2.3.1 definitions and general provisions2.3.2 assignment of packing group2.3.3 determination of flashpoint2.3.4 determination of initial boiling pointChapter 2.4 class 4 – flammable solids; substances liable to spontaneous combustion; substances which, in contact with water, emit flammable gases2.4.0 introductory note2.4.1 definition and general provisions2.4.2 class 4.1 – flammable solids, self-reactive substances and solid desensitized explosives 2.4.3 class 4.2 – substances liable to spontaneous combustion2.4.4 class 4.3 – substances which, in contact with water, emit flammable gases2.4.5 classification of organometallic substancesChapter 2.5 class – oxidizing substances and organic peroxides2.5.0 introductory note2.5.1 definitions and general provisions2.5.2 class 5.1 – oxidizing substances2.5.3 class 5.2 – organic peroxidesChapter 2.6 class 6 – toxic and infectious substances2.6.0 introductory notes2.6.1 definitions2.6.2 class 6.1 – toxic substances2.6.3 class 6.2 – infectious substancesChapter 2.7 class 7 – radioactive material2.7.1 definitions2.7.2 classificationChapter 2.8 class 8 – corrosive substances2.8.1 definitions and properties2.8.2 assignment of packing groupsChapter 2.9 miscellaneous dangerous substances and articles (class 9) and environmentally hazardous substances2.9.1 definitions2.9.2 assignment to class 92.9.3 environmentally hazardous substances (aquatic environment)Chapter 2.10 marine pollutants2.10.1 definition2.10.2 general provisions2.10.3 classificationPart 3 – dangerous goods list, special provisions and exceptions~ ···········Part 4 – packing and tank provisionsChapter 4.1 use of packaging, including intermediate bulk containers (IBCs) and large packaging4.1.0 definitions4.1.1 general provisions for the packing of dangerous goods in packings, including IBCs and large packaging4.1.2 additional general provisions for the use of IBCs4.1.3 general provisions concerning packing instructions4.1.4 list of packing instructionspacking instructions concerning the use of packaging (except IBCs and large packaging)packing instructions concerning the use of IBCspacking instructions concerning the use of large packaging4.1.5 special packing provisions for goods of class 14.1.6 special packing provisions for goods of class 24.1.7 special packing provisions for organic peroxides (class5.2) and self-reactive substances of class 4.14.1.8 special packing provisions for infectious substances of category A (class 6.2, UN 2814 and UN 2900)4.1.9 special packing provisions for class 7。

VDA Structure Component Requirement SpecificationModule ICRS Universal Requirements<Title><Number>Version 1.0ReleaseTechnical depart-ment/functionName Date SignatureCRS authorTechnical departmentProject managementDevelopmentTable of Contents0Change History 8-4 1Foreword 8-5 2General Project Guidelines 8-6 2.1Objective 8-6 2.2Organisation 8-6 2.3Sub-Supplier Management 8-6 2.4Production Process Release and Product Release 8-7 2.5Performance Specification 8-7 2.6Variant Management 8-7 2.7Test Management 8-8 2.8Change Management 8-8 2.9General Development and Supply Scope 8-8 2.10Content Amendments 8-8 3Scheduling in the Project 8-9 4Reporting and Documentation 8-10 4.1Information Exchange 8-10 4.2Documentation and Archiving Obligations 8-10 4.3Priority and Up-to-Dateness of Documents 8-10 4.4Change Documentation and Part History Documentation 8-11 4.5Vehicle Documentation 8-11 4.6Traceability 8-11 4.7Labelling of Parts 8-11 4.8Content Amendments 8-12 5Quality Requirements on the Product Design 8-13 5.1Quality Contribution 8-13 5.2Quality Management System 8-13 5.3Preventive QM Methods and Safeguarding Against Risk 8-13 5.4Maturity Level Assurance in the Supply Chain 8-13 5.5Requalification 8-14 5.6Complaint Processing 8-14 5.7Error Management 8-14 5.8Content Amendments 8-14 6Nondisclosure 8-157Product Data Management 8-16 7.1CAx and EDM Supplier Integration 8-16 7.1.1Qualification of the Suppliers 8-16 7.1.2Data Exchange Agreement 8-16 7.2CAD Requirements 8-16 7.3DMU Requirements 8-17 7.4Parts List and Documentation Method 8-17 7.5Reference Point System and Functional Dimensions 8-17 7.6Requirements on the Creation of Drawings 8-17 7.7Content Amendments 8-17 8Environmental Compatibility 8-18 8.1Recycling Scheme 8-18 8.2Environmental Characteristics of the Materials 8-18 8.3Dismantling Scheme 8-18 8.4Content Amendments 8-18 9Material Specification and Surfaces 8-19 9.1Material Directives and Prohibitions 8-19 9.2Universal Material Requirements 8-19 9.3Requirements on the Weather Resistance 8-19 9.4Requirements on Electrical Components 8-19 9.5Surface Protection, Surfaces, Edges 8-20 9.6Corrosion Protection 8-20 10Logistic Requirements 8-21 11Customer Service and other Service Requirements 8-22 12Requirements on Test and Prototype Tools 8-23 13Standard and Repeat Parts 8-24 14Content Amendments to Module I 8-25 15Definitions, Terms, Acronyms 8-26 16Further Applicable Documents 8-270 Change HistoryEdited (date) Approved by No. Amendment DescriptionOEM OEM1 ForewordEditor comments (delete in the final version)This chapter is an informative introductory element that gives certain information or commentaries about the purpose of the requirementspecification (e.g. that the requirement specification describes services, requirements, inspection and testing conditions that the product to be developed has to fulfil). The foreword must not contain any technical or functional requirements, images or tables.Rules for handling the VDA component requirement specification structure1. Structure: level 1 and level 2 of the VDA component requirementspecification structure have to be kept and must not be changed.2. Expansion to the structure: if amendments are necessary in theimplementation of the VDA structure, in each case these are to be added to the last chapter with content of Module I or Module II (see Module I, chapter 12 and Module II chapter7).Expansions of the level 2 of the VDA structure are to be placed at the end of a respective sub-chapter structure (see e.g. Module I,chapter 2.6).3. Headlines that are not used may not be deleted on levels 1 and 2 ofthe Modules I and II, in order to guarantee the transparency and clarity of the component requirement specification contents. Instead, thesechapters are to be marked with the reference “not relevant for thiscomponent”.When referencing an external document it has to be entered here.4. Contents on level 3 are to be preferentially used and have the natureof a recommendation. If chapters from level 3 are not used, these may be deleted or renamed.This document is based on the VDA CRS structure. It consists of universal requirements for the component requirement specification (Module I) and the component requirement specification for mechanical and E/E components (Module II). For more information please go to www.vda-qmc.de.2 General Project Guidelines2.1 ObjectiveEditor comments (delete in the final version)The superordinate objectives are to be defined in thischapter (development, production, etc). The component-specificobjectives that go beyond this are to be defined in Module II.2.2 OrganisationEditor comments (delete in the final version)The fundamental, project-wide organisational general conditions are to be defined in this chapter (e.g. committee, control processes, project management plan).These especially include measures for project controlling and theestablishing of development-accompanying reviews and information about risk management.Furthermore, standards (minimum content, interface characteristics or tools to be used, formal layout, etc.) for the documentation of recurrent events along the course of the project (e.g. meeting minutes, status or progress reports and reviews, end of project documentation, etc.) are to be defined in this chapter.2.3 Sub-Supplier ManagementEditor comments (delete in the final version)Stipulations concerning the sub-suppliers that are to be involved by the contractor can be made here (e.g. duty of notification about theinvolvement of sub-suppliers, customer requirements on contractsbetween the contractors, deadline responsibilities, non-disclosureagreements, data formats, documentation).Requirements on the contractor or sub-supplier can correspond to,among other things, the project structure, capacity requirements,communication or auditing duties (proof of qualification)Furthermore, stipulations can be made here that result from the defined duties of the contractor from the chapter “Responsibilities” (e.g. forpartial scopes of a component that are to be developed and produced by the sub-supplier).2.4 Production Process Release and Product ReleaseEditor comments (delete in the final version)The relevant processes for the acceptance of components, systems or patterns are to be described in this chapter. The contractor’s duty to co-operate is to be differentiatedly described. If available, reference should be made to the customer’s existing incoming goods conditions.Furthermore, the relevant rights and obligations of the inspection and release processes for the contractor are to be described here.2.5 Performance SpecificationEditor comments (delete in the final version)In this chapter it is to be agreed how and with what contents thecontractor is to generate a performance specification. For the fulfilment of all requirements, especially with regard to durability, properties and condition, the contractor has to comment in writing in the performance specification.2.6 Variant ManagementEditor comments (delete in the final version)General requirements on the variant management are to be defined in this chapter. Furthermore, it is to be recorded here how additionalvariants are to be handled or how and in which systems variants are to be documented.2.7 Test ManagementEditor comments (delete in the final version)General requirements and stipulations on tests are to be defined in this chapter (e.g. test planning and documentation, etc).2.8 Change ManagementEditor comments (delete in the final version)In this chapter criteria and workflows are to be established for how to, for example, coordinate, approve and document cost, weight and deadline-relevant changes during the development time.2.9 General Development and Supply ScopeEditor comments (delete in the final version)All general requirements on the development and delivery scopes are to be defined in this chapter. This includes all services necessary for the fulfilment of the requirements.2.10 Content AmendmentsEditor comments (delete in the final version)If the specified content structure is not sufficient to assign all require-ments on level 2 to the superordinate subject on level 1, the require-ments can be assigned to this chapter.If this chapter is not necessary, you should delete it!3 Scheduling in the ProjectEditor comments (delete in the final version)Project-specific deadlines that are important for the contractor andcustomer in the development and, if necessary, in the series production of the component are to be defined and communicated in this chapter (to some extent the deadlines themselves are derived from thesuperordinated plans [model politics]).Furthermore, a definition of project-specific milestones or productmaturity level grades that are to be observed by the contractor during the course of the project is to be made. Reference to existing standards is to be made in the definition of the product maturity level grades, if possible.Additionally, metrics for project tracking with which the customer checks the project progress can be listed. Typical metrics are for examplemilestone trend analysis, expense or error tracking. The data for themetrics to be transferred by the contractor and the intervals of the data transfer are established here.4 Reporting and Documentation4.1 Information ExchangeEditor comments (delete in the final version)The mutual information obligations and the project-wide standarddocumentations (e.g. meeting minutes, testing documentation, technical [interim] and status reports, work reports, to-do lists, FMEA reports, FTA reports) are to be defined in this chapter.In addition to formal guidelines, reference is to be made in particular to the principal responsibilities or the obligations to co-operate of thecontractor and customer in the reporting along the development process.4.2 Documentation and Archiving ObligationsEditor comments (delete in the final version)The determination of project-wide retention and archiving periods is to be made taking existing cross-manufacturer standards (e.g. VDA volume 1) into consideration. In addition to the definition of criteria and processes for archiving, the data processing systems required by the contractor are also to be established.4.3 Priority and Up-to-Dateness of DocumentsEditor comments (delete in the final version)Suitable rules are to be defined as to how the contractor and customer are to react in the case of contradictions (e.g. in CRS Module II).Additionally, priority and up-to-dateness rules for different documents are to be defined in this chapter.4.4 Change Documentation and Parts History DocumentationEditor comments (delete in the final version)In this chapter the customer defines the data and its form of recording that are to be made available by the contractor in the framework of a change documentation in the course of the development and, ifapplicable, production processes (e.g. entries in a project manual).4.5 Vehicle DocumentationEditor comments (delete in the final version)In this chapter all parts for which there are special documentationrequirements due to safety or certification relevance are to be listed (see also VDA volume 1 – determination of documents with special archiving).The contractor’s duty to co-operate and the data processing system to be used are also to be established.4.6 TraceabilityEditor comments (delete in the final version)A component-specific determination of processes, procedures and dataprocessing system that ensure the traceability (e.g. part batch) is to be safeguarded here.1.7 Labelling of PartsEditor comments (delete in the final version)The customer requirements on various types of labelling (e.g. for series and original parts, test and prototype parts) is to be established in this chapter.4.8 Content AmendmentsEditor comments (delete in the final version)If the given content structure does not suffice to assign all requirements on level 2 to the superordinated subject on level 1, the requirements can be assigned to this chapter.If this chapter is not needed, you should delete it!5 Quality Requirements on the Product Design5.1 Quality ContributionEditor comments (delete in the final version)In this chapter, general requirements on the contractor concerning its quality assurance (factory, project) are to be defined (e.g. quality testing of prototype, pre-series and series production parts).The OEM-specific Q-guidelines also have to be stated here.5.2 Quality Management SystemEditor comments (delete in the final version)The requirements on the contractor’s QM system (including interfaces to the customer and sub-suppliers) are to be defined here; reference to existing standards if possible (e.g. ISO/TS 16949).5.3 Preventive QM Methods and Safeguarding against RiskEditor comments (delete in the final version)Here the fundamental measures for the concept, product and process safeguarding (e.g. quality functional deployment, FMEA, system, design or process FMEAs, reviews) that are valid for all awardings within aproject are to be requested by the customer.5.4 Maturity Level Assurance in the Supply ChainEditor comments (delete in the final version)Which quality-relevant processes for the maturity level assurance in the supply chain are to be documented and assured at the contractor’s are to be defined in this chapter.5.5 RequalificationEditor comments (delete in the final version)The contractor is to be strictly committed to the requalification test (RQT) in series production. The customer is to bindingly define the objectives, content, processes and cycles of the RQT here.5.6 Complaint ProcessingEditor comments (delete in the final version)In this chapter the requirements of how the contractor is to deal with remedial actions and downstream efficiency examinations within the framework of complaint processing are to be defined.5.7 Error ManagementEditor comments (delete in the final version)Requirements on the contractor’s and the customer’s error management can be defined here (error documentation, error analysis, error remedial actions, efficiency test, etc).5.8 Content AmendmentsEditor comments (delete in the final version)If the given content structure does not suffice to assign all requirements on level 2 to the superordinated subject on level 1, the requirements can be assigned to this chapter.If this chapter is not needed, you should delete it!6 Non-DisclosureEditor comments (delete in the final version)In this chapter the general rights and obligations of the contractor and customer to non-disclosure, reuse, evaluation, etc. of the information contained in the component requirement specification can be defined by the customer.7 Product Data Management7.1 CAx and EDM Supplier Integration7.1.1 Qualification of the SuppliersEditor comments (delete in the final version)In this sub-chapter, the qualification requirements on the contractor or sub-supplier and/or their employees with regard to CAx and EDM are to be listed. If available, a reference should be made to the trainingopportunities offered by the customer.7.1.2 Data Exchange AgreementEditor comments (delete in the final version)In this chapter, the demands for digital safeguarding of the development as well as concerning minimum standards in regard to data exchange formats (e.g. CATIA V.x, PRO ENGINEER V.x, 3D CAD data) are to be defined.The minimum requirements concerning the electronic data exchange between contractor and customer should define not only the reference to all necessary forms but also the differentiated layout of geometricaldescriptions, CAD datasets and data transfers (e.g. RDT standards, online access to engineering portals).If available, reference should be made to any existing cross-manufacturer standards (e.g. VDA volume 4.2).7.2 CAD RequirementsEditor comments (delete in the final version)Describe the component-specific 3D CAD data that is made available by the contractor and the customer (e.g. cross sections, views).The contractor’s obligations in data cleaning or checking, geometricdescription, the CAD data quality and DMU requirements are also to be documented here.7.3 DMU RequirementsEditor comments (delete in the final version)In this chapter the customer’s general requirements on the Digital Mock-Up (DMU) are to be formulated (if necessary with reference to existing regulations, rules, etc.)7.4 Parts List and Documentation MethodEditor comments (delete in the final version)Define the requirements on the contents and the form of the parts list to be kept by the contractor (if the contractor is responsible for keeping the parts list).7.5 Reference Point System and Functional DimensionEditor comments (delete in the final version)Determine the necessary nomenclature and the most importantrequirements on the reference point system to be kept by the contractor.7.6 Requirements on the Creation of DrawingsEditor comments (delete in the final version)Define the requirements on the content and form of the drawings.Especially the references to the customer’s respectively valid rules with regard to the creation of drawings are to be entered here.7.7 Content AmendmentsEditor comments (delete in the final version)If the given content structure does not suffice to assign all requirements on level 2 to the superordinated subject on level 1, the requirements can be assigned to this chapter.If this chapter is not needed, you should delete it!8 Environmental Compatibility8.1 Recycling SchemeEditor comments (delete in the final version)This chapter establishes the general project-wide requirements on the recycling and repair scheme in the development of a component. Ifpossible, this chapter is also to make reference to existing laws and standards (cross-manufacturer), (e.g. VDA volume 31, VDA 260[labelling of materials], directive 2000/53 EC [End of Life Vehicles] and 761/2001 EC [EMAS]).8.2 Environmental Characteristics of the MaterialsEditor comments (delete in the final version)In this chapter the cross-component requirements in regard to permitted and forbidden (e.g. harmful to health) materials (e.g. lead-free solder).8.3 Dismantling SchemeEditor comments (delete in the final version)The dismantling scheme describes the requirements on the contractor in terms of a simple, unmixed dismantling of the component as well as its accessibility when assembled.8.4 Content AmendmentsEditor comments (delete in the final version)If the given content structure does not suffice to assign all requirements on level 2 to the superordinated subject on level 1, the requirements can be assigned to this chapter.If this chapter is not needed, you should delete it!9 Material Specification and Surfaces9.1 Material Directives and ProhibitionsEditor comments (delete in the final version)In this chapter the cross-component requirements on the materials to be used or the materials not to be used are to be defined. These areproject-wide regulations in regard to mandatory materials or theircombinations.9.2 Universal Material RequirementsEditor comments (delete in the final version)In this chapter the universal requirements on the materials themselves, the determination of materials, scope of the material determination, as well as the determinations for material specifications etc. are to bespecified.The general material requirements include requirements on theinflammability of materials, their compatibility, emissions behaviour, etc.9.3 Requirements on the Weather ResistanceEditor comments (delete in the final version)In this chapter the general requirements on the weather resistance of materials and the proof of this are to be determined.9.4 Requirements on Electric ComponentsEditor comments (delete in the final version)In addition to the general requirements on the materials, in this chapter especially the material requirements for electric and electronic parts and components are to be defined.9.5 Surface Protection, Surfaces, EdgesEditor comments (delete in the final version)In this chapter the general requirements and regulations for surface pro-tection and the properties and condition of the surfaces and edges is to be determined.9.6 Corrosion ProtectionEditor comments (delete in the final version)In this chapter the customer’s universal requirements for corrosionprotection are to be described.These include perforation corrosion requirements and/or guarantees, especially in the chassis area and the body, as well as requirements in regard to contact, surface, crevice, cavity, stress and filiform corrosions, etc.10 Logistics RequirementsEditor comments (delete in the final version)In this chapter the customer can make determinations to the logistic scheme that are valid for all components of a project. These include e.g.universal requirements on the supply of individual production sites or standards regarding packaging, carriers, etc. If available, reference is to be made to the additional standards of the customer.11 Customer Service and other Service RequirementsEditor comments (delete in the final version)In this chapter the requirements on the replacement part documentation, such as replacement part catalogue, replacement wear out lists,drawings, isometric replacement part depictions in electronic form, etc., that the customer needs from the contractor are to be determined. The time of provision before the start of series production is to be stated.12 Requirements on Test and Prototype ToolsEditor comments (delete in the final version)In this chapter the general requirements on the handling of test and prototype tools is to be defined (commissioning, development,manufacturing of test parts from prototype tools, costs, disposal, etc).13 Standard and Repeat PartsEditor comments (delete in the final version)The customer-specific requirements regarding installation quota and use of standard, repeat and same parts are to be defined here. It should be observed that all relevant information in terms of standard, repeat and same parts is to be made available to the contractor.14 Content Amendments to Module IEditor comments (delete in the final version)If the given content structure does not suffice to assign all requirements on level 1, the requirements can be assigned to this chapter.If this chapter is not needed, you should delete it!15 Definitions, Terms, AcronymsEditor comments (delete in the final version)The table of acronyms is to be supplemented by a standardised glossary in which the most important terms that are used within the requirement specification are uniformly defined. For this, the same terms from other VDA volumes are to be used for the same facts.CAx Computer Aided (x = variable)CPM Critical Parts ManagementCRS Module I Component Requirement Specification Module I CRS Module II Component Requirement Specification Module II DMU Digital Mock-UpDP Data ProcessingEDM Engineering Data ManagementE/E Electrical/ElectronicEMAS Environmental Management and Auditing Scheme FMEA Failure Mode and Effects AnalysisFTA Fault Tree AnalysisIP Ingress ProtectionISO International Standards OrganisationMMI Multimedia InterfaceOEM Original Equipment ManufacturerQM Quality ManagementRDT Remote Data TransmissionRQT Requalification TestSW SoftwareTS Technical Specification16 Further Applicable DocumentsEditor comments (delete in the final version)In this connection, only the documents quoted from in the requirement specification are to be considered further applicable documents.The further applicable documents (laws, standards, customer-specific rules, etc.) that are referred to in the requirement specification textshould be differentiatedly listed according to type of document.Furthermore, a source (alternative: contact person) of the information for the contractor is to be named.The further applicable documents that were valid at the CRS issue date are applicable.If reference is made to regulations (laws, ordinances and such), thereference has to be in such a manner that the procurement is possible.The access address and modalities for the supplier are also to be named here.。

EUROPEAN PHARMACOPOEIA 5.0 2.6.14.BacterialendotoxinsMaize starch 1.0g Sodium chloride5.0gAgar,according to gelling power 10.0g to 15.0gPurified water1000mlHydrate the agar,dissolve by heating to boiling with continuous stirring.If necessary,adjust the pH so that after sterilisation it is 7.3±0.2.Sterilise by heating in an autoclave at 121°C for 15min.Allow to cool to 45-50°C;add,where necessary,gentamicin sulphate corresponding to 20mg of gentamicin base and pour into Petri dishes.Medium R (Lactose monohydrate sulphite medium)Pancreatic digest of casein 5.0g Yeast extract 2.5g Sodium chloride 2.5g Lactose monohydrate 10.0g Cysteine hydrochloride 0.3g Purified water1000mlDissolve,adjust to pH 7.1±0.1and fill to 8ml in16mm ×160mm tubes containing a small Durham tube.Sterilise by heating in an autoclave at 121°C for 15min and store at 4°C.Before use,heat the medium for 5min in a water-bath and cool.Add to each tube 0.5ml of a 12g/l solution of sodium metabisulphite R and 0.5ml of a 10g/l solution of ferric ammonium citrate,both solutions being freshly preparedand filtered through membranes (pore size:0.45µm).Agar medium S (R2A)Yeast extract 0.5g Proteose peptone 0.5gCasein hydrolysate0.5g Glucose0.5g Starch0.5g Dipotassium hydrogen phosphate0.3g Magnesium sulphate,anhydrous0.024g Sodium pyruvate 0.3g Agar 15.0g Purified water1000mlAdjust the pH so that after sterilisation it is 7.2±0.2.Sterilise by heating in an autoclave at 121°C for 15min.NEUTRALISING AGENTSNeutralising agents may be used to neutralise the activity of antimicrobial agents.They may be added to buffered sodium chloride-peptone solution pH 7.0,preferably before sterilisation.If utilised their efficacy and non-toxicity towards micro-organisms are demonstrated.A typical neutralising fluid has the following composition:Polysorbate 8030g Lecithin (egg)3g Histidine hydrochloride 1g Peptone (meat or casein)1g Sodium chloride4.3g Potassium dihydrogen phosphate 3.6g Disodium hydrogen phosphate dihydrate 7.2g Purified water1000mlSterilise by heating in an autoclave at 121°C for 15min.If the solution has insufficient neutralising capacity the concentration of polysorbate 80or lecithin may be increased.Alternatively,the neutralisers mentioned in Table 2.6.13.-3may be added.Table 2.6.13.-3.–Inactivators for antimicrobial agents to be added to buffered sodium chloride-peptone solution pH 7.0Type of an-timicrobial agentInactivator Concentration Comment Sodium laurilsulfate 4g/l Polysorbate 80and lecithin 30g/l and 3g/l Phenolics Egg yolk 5ml/l -50ml/l Add after sterilisation of buffered sodium chloride-peptone solution pH 7.0Organo-mercurals Sodium thioglycolate 0.5g/l -5g/lHalogens Sodium thiosulphate 5g/l Quaternaryammonium compounds Egg yolk5ml/l -50ml/lAdd after sterilisation of buffered sodium chloride-peptonesolution pH 7.001/2005:206142.6.14.BACTERIAL ENDOTOXINS The test for bacterial endotoxins is used to detect or quantify endotoxins of gram-negative bacterial originusing amoebocyte lysate from horseshoe crab (Limuluspolyphemus or Tachypleus tridentatus ).There are 3techniques for this test:the gel-clot technique,which is based on gel formation;the turbidimetric technique,based on the development of turbidity after cleavage of an endogenous substrate;and the chromogenic technique,based on the development of colour after cleavage of a synthetic peptide-chromogen complex.The following 6methods are described in the present chapter:Method A.Gel-clot method:limit testMethod B.Gel-clot method:semi-quantitative test Method C.Turbidimetric kinetic method Method D.Chromogenic kinetic method Method E.Chromogenic end-point method Method F.Turbidimetric end-point methodProceed by any of the 6methods for the test.In the eventof doubt or dispute,the final decision is made based upon method A unless otherwise indicated in the monograph.The test is carried out in a manner that avoids endotoxin contamination.ApparatusDepyrogenate all glassware and other heat-stable apparatus in a hot-air oven using a validated process.A commonly used minimum time and temperature is 30minutes at 250°C.If employing plastic apparatus,such as microtitre plates and pipette tips for automatic pipetters,use apparatus shown to be free of detectable endotoxin and of interfering effects for the test.NOTE:In this chapter,the term ‘tube’includes all types of receptacles,for example microtitre plate wells .2.6.14.Bacterial endotoxins EUROPEAN PHARMACOPOEIA5.0Preparation of the standard endotoxin stock solutionThe standard endotoxin stock solution is prepared froman endotoxin reference standard that has been calibrated against the International Standard,for example endotoxin standard BRP.Endotoxin is expressed in International Units(IU).The equivalence in IU of the International Standard is stated by the World Health Organisation.NOTE:One International Unit(IU)of endotoxin is equal to one Endotoxin Unit(E.U.).Follow the specifications in the package leaflet and on the label for preparation and storage of the standard endotoxin stock solution.Preparation of the standard endotoxin solutionsAfter vigorously mixing the standard endotoxin stock solution,prepare appropriate serial dilutions of this solution using water for bacterial endotoxins test(water for BET). Use the solutions as soon as possible to avoid loss of activity by adsorption.Preparation of the test solutionsPrepare the test solutions by dissolving or diluting active substances or medicinal products using water for BET. Some substances or preparations may be more appropriately dissolved or diluted in other aqueous solutions.If necessary, adjust the pH of the test solution(or dilution thereof)so that the pH of the mixture of the lysate and test solution falls within the pH range specified by the lysate manufacturer. This usually applies to a product with a pH in the range of6.0 to8.0.The pH may be adjusted by the use of acid,base or a suitable buffer,as recommended by the lysate manufacturer. Acids and bases may be prepared from concentrates or solids with water for BET in containers free of detectable endotoxin.Buffers must be validated to be free of detectable endotoxin and interfering factors.Determination of the Maximum Valid DilutionThe Maximum Valid Dilution(MVD)is the maximum allowable dilution of a sample at which the endotoxin limit can be determined.Determine the MVD using the following formulae:Endotoxin limit:the endotoxin limit for active substances administered parenterally,defined on the basis of dose,is equal to:K=threshold pyrogenic dose of endotoxin perkilogram of body mass in a single hour period,M=maximum recommended dose of product perkilogram of body mass in a single hour period. The endotoxin limit for active substancesadministered parenterally is specified in units suchas IU/ml,IU/mg,IU/Unit of biological activity,etc.,in monographs.Concentration of test solution:—in mg/ml if the endotoxin limit is specified by mass (IU/mg),—in Units/ml if the endotoxin limit is specified by unit of biological activity(IU/Unit),—in ml/ml if the endotoxin limit is specified by volume (IU/ml).λ=the labelled lysate sensitivity in the gel-clottechnique(IU/ml)or the lowest point usedin the standard curve of the turbidimetric orchromogenic techniques.GEL-CLOT TECHNIQUE(METHODS A AND B)The gel-clot technique allows detection or quantificationof endotoxins and is based on clotting of the lysate in the presence of endotoxins.The concentration of endotoxins required to cause the lysate to clot under standard conditions is the labelled lysate sensitivity.To ensure both the precision and validity of the test,confirm the labelled lysate sensitivity and perform the test for interfering factors as described under1.Preparatory testing.1.PREPARATORY TESTING(i)Confirmation of the labelled lysate sensitivity Confirm in4replicates the labelled sensitivityλ,expressed in IU/ml,of the lysate solution prior to use in the test. Confirmation of the lysate sensitivity is carried out when a new batch of lysate is used or when there is any change in the experimental conditions which may affect the outcome of the test.Prepare standard solutions of at least4concentrations equivalent to2λ,λ,0.5λand0.25λby diluting the standard endotoxin stock solution with water for BET.Mix a volume of the lysate solution with an equal volumeof1of the standard solutions(such as0.1ml aliquots)in each tube.When single test vials or ampoules containing lyophilised lysate are employed,add solutions directly to the vial or ampoule.Incubate the reaction mixture for a constant period according to the recommendations of the lysate manufacturer(usually at37±1°C for60±2min), avoiding vibration.Test the integrity of the gel:for tubes, take each tube in turn directly from the incubator and invert it through approximately180°in one smooth motion.If a firm gel has formed that remains in place upon inversion, record the result as positive.A result is negative if an intact gel is not formed.The test is not valid unless the lowest concentration of the standard solutions shows a negative result in all replicate tests.The end-point is the last positive result in the series of decreasing concentrations of endotoxin.Calculate the mean value of the logarithms of the end-point concentrations and then the antilogarithm of the mean value using the following expression:Geometric mean end-point concentration==sum of the log end-point concentrations of the dilution series used,f=number of replicates.The geometric mean end-point concentration is the measured sensitivity of the lysate solution(IU/ml).If this is not less than0.5λand not more than2λ,the labelled sensitivity is confirmed and is used in the tests performed with this lysate. (ii)Test for interfering factorsPrepare solutions A,B,C and D as shown in Table2.6.14.-1, and use the test solutions at a dilution less than the MVD, not containing any detectable endotoxins,operating as described under1.Preparatory testing,(i)Confirmation of the labelled lysate sensitivity.EUROPEAN PHARMACOPOEIA 5.0 2.6.14.BacterialendotoxinsTable 2.6.14.–1Solution Endotoxin concentration/Solution towhich endotoxin is addedDiluent Dilution factorInitial endotoxin concentrationNumber of replicatesA None/Test solution ---4B2λ/Test solutionTest solution12482λ1λ0.5λ0.25λ4444C 2λ/Water for BET Water for BET12482λ1λ0.5λ0.25λ2222D None/Water for BET---2Solution A =solution of the preparation being examined that is free of detectable endotoxins.Solution B =test for interference.Solution C =control of the labelled lysate sensitivity.Solution D =negative control (water for BET).The geometric mean end-point concentrations of solutions B and C are determined using the expression described in 1.Preparatory testing,(i)Confirmation of the labelled lysate sensitivity.The test for interfering factors is repeated when any changes are made to the experimental conditions that are likely to influence the result of the test.The test is not valid unless all replicates of solutions A and D show no reaction and the result of solution C confirms the labelled lysate sensitivity.If the sensitivity of the lysate determined with solution B is not less than 0.5λand not greater than 2λ,the test solution does not contain interfering factors under the experimental conditions used.Otherwise,the solution interferes with the test.If the preparation being examined interferes with the test at a dilution less than the MVD,repeat the test for interfering factors using a greater dilution,not exceeding the MVD.The use of a more sensitive lysate permits a greater dilution of the preparation being examined and this may contribute to the elimination of interference.Interference may be overcome by suitable treatment,such as filtration,neutralisation,dialysis or heat treatment.To establish that the treatment chosen effectively eliminates interference without loss of endotoxins,repeat the test for interfering factors using the preparation being examined to which the standard endotoxin has been added and which has then been submitted to the chosen treatment.2.LIMIT TEST (METHOD A)(i)ProcedurePrepare solutions A,B,C and D as shown in Table 2.6.14.-2,and perform the test on these solutions following the procedure described under 1.Preparatory testing,(i)Confirmation of the labelled lysate sensitivity.Table 2.6.14.-2Solution Endotoxin concentration/Solution to which endotoxin is addedNumber of replicates A None/Diluted test solution2B 2λ/Diluted test solution 2C 2λ/Water for BET2D None/Water for BET2Prepare solution A and solution B (positive product control)using a dilution not greater than the MVD and treatments as described in 1.Preparatory testing,(ii)Test for interferingfactors.Solutions B and C (positive controls)contain the standard endotoxin at a concentration corresponding to twice the labelled lysate sensitivity.Solution D (negative control)consists of water for BET.(ii)InterpretationThe test is not valid unless both replicates of the 2positive control solutions B and C are positive and those of the negative control solution D are negative.The preparation being examined complies with the test when a negative result is found for both replicates of solution A.When a positive result is found for both replicates of solution A:—if the preparation being examined is diluted to the MVD,it does not comply with the test,—if the preparation being examined is diluted to a dilution less than the MVD,the test is repeated at a dilution not greater than the MVD.Repeat the test if a positive result is found for one replicate of solution A and a negative result is found for the other.The preparation being examined complies with the test if a negative result is found for both replicates of solution A in the repeat test.3.SEMI-QUANTITATIVE TEST (METHOD B)(i)ProcedureThe test quantifies bacterial endotoxins in the test solution by titration to an end-point.Prepare solutions A,B,C and D as shown in Table 2.6.14.-3,and test these solutions according to the procedure described under 1.Preparatory testing,(i)Confirmation of the labelled lysate sensitivity.(ii)Calculation and interpretationThe test is not valid unless the following 3conditions are met:(a)both replicates of solution D (negative control)are negative,(b)both replicates of solution B (positive product control)are positive,(c)the geometric mean end-point concentration of solution Cis in the range of 0.5λto 2λ.To determine the endotoxin concentration of solution A,calculate the end-point concentration for each replicate series of dilutions by multiplying each end-point dilutionfactor by λ.The endotoxin concentration in the test solution is the geometric mean end-point concentration of the replicates(see the expression given under 1.Preparatory testing,(i)Confirmation of the labelled lysate sensitivity).If the2.6.14.Bacterial endotoxins EUROPEAN PHARMACOPOEIA5.0test is conducted with a diluted test solution,calculatethe concentration of endotoxin in the original solution by multiplying the result by the dilution factor.If none of the dilutions of the test solution is positive in a valid test,record the endotoxin concentration as less thanλ(or,if a diluted sample was tested,as less thanλ×the lowest dilution factor of the sample).If all dilutions are positive,the endotoxin concentration is recorded as equal to or greater than the greatest dilution factor multiplied byλ(e.g.in Table2.6.14.-3,the initial dilution factor×8×λ).The preparation meets the requirements of the test if the endotoxin concentration is less than that specified in the individual monograph.PHOTOMETRIC TECHNIQUES(METHODS C,D,E AND F) 1.TURBIDIMETRIC TECHNIQUE(METHODS C AND F) This technique is a photometric test to measure the increase in turbidity.Based on the test principle employed,this technique is classified as being the end-point-turbidimetric test or the kinetic-turbidimetric test.The end-point-turbidimetric test(Method F)is basedon the quantitative relationship between the endotoxin concentration and the turbidity(absorbance or transmission) of the reaction mixture at the end of an incubation period. The kinetic-turbidimetric test(Method C)is a method to measure either the time(onset time)needed for the reaction mixture to reach a predetermined absorbance,or the rate of turbidity development.The test is carried out at the incubation temperature recommended by the lysate manufacturer(usually37±1°C).2.CHROMOGENIC TECHNIQUE(METHODS D AND E) This technique is used to measure the chromophore released from a suitable chromogenic peptide by the reactionof endotoxins with the lysate.Depending on the test principle employed,this technique is classified as being the end-point-chromogenic test or the kinetic-chromogenic test.The end-point-chromogenic test(Method E)is basedon the quantitative relationship between the endotoxin concentration and the quantity of chromophore released at the end of an incubation period.The kinetic-chromogenic test(Method D)measures either the time(onset time)needed for the reaction mixture to reach a predetermined absorbance,or the rate of colour development.The test is carried out at the incubation temperature recommended by the lysate manufacturer(usually37±1°C).3.PREPARATORY TESTINGTo assure the precision or validity of the turbidimetric and chromogenic tests,preparatory tests are conducted to assure that the criteria for the standard curve are satisfied and that the test solution does not interfere with the test. Validation of the test method is required when any changes are made to the experimental conditions that are likely to influence the result of the test.(i)Assurance of criteria for the standard curveUsing the standard endotoxin solution,prepare at least3endotoxin concentrations to generate the standard curve.Perform the test using at least3replicates of each standard endotoxin solution as recommended by the lysate manufacturer(volume ratios,incubation time,temperature, pH,etc.).If the desired range is greater than2log in the kinetic methods,additional standards must be included to bracket each log increase in the range of the standard curve.The absolute value of the correlation coefficient,|r|,must be greater than or equal to0.980,for the range of endotoxin concentrations indicated by the lysate manufacturer.(ii)Test for interfering factorsSelect an endotoxin concentration at or near the middle of the endotoxin standard curve.Prepare solutions A,B,C and D as shown in Table2.6.14.-4. Perform the test on at least2replicates of these solutions as recommended by the lysate manufacturer(volume of test solution and lysate solution,volume ratio of test solution to lysate solution,incubation time,etc.).Table2.6.14.-3Solution Endotoxin concentration/Solution towhich endotoxin is added Diluent Dilution factor Initial endotoxinconcentrationNumber of replicatesA None/Test solution Water for BET1248----2222B2λ/Test solution12λ2C2λ/Water for BET Water for BET12482λ1λ0.5λ0.25λ2222D None/Water for BET---2 Solution A=test solution at the dilution,not exceeding the MVD,with which the test for interfering factors was carried out.Subsequent dilution of the test solution must not exceed the e water for BET to make two dilution series of1,1/2,1/4and1/8,relative to the dilution with which the test for interfering factors was carried out.Other dilutions may be used as appropriate.Solution B=solution A containing standard endotoxin at a concentration of2λ(positive product control).Solution C=2series of water for BET containing the standard endotoxin at concentrations of2λ,λ,0.5λand0.25λ.Solution D=water for BET(negative control).EUROPEAN PHARMACOPOEIA 5.0 2.6.14.BacterialendotoxinsTable 2.6.14.-4.Solution Endotoxin concentrationSolution to which endotoxin is added Number of replicates A NoneTest solution Not less than 2B Middle concentration of the standard curve Test solution Not less than 2CAt least 3concentra-tions (lowest concen-tration is designated λ)Water for BETEach concentra-tion not less than 2D NoneWater for BETNot less than 2Solution A =test solution,that may be diluted not to exceed the MVD.Solution B =preparation to be examined at the same dilution assolution A,containing added endotoxin at a concentration equal to or near the middle of the standard curve.Solution C =standard endotoxin solution at the concentrations used in the validation of the method as described under 3.Preparatory testing,(i)Assurance of criteria for the standard curve (positive controls.Solution D =water for BET (negative control).Calculate the mean recovery of the added endotoxin by subtracting the mean endotoxin concentration in the solution (if any)from that in the solution containing the added endotoxin.The test solution is considered free of interferingfactors if under the conditions of the test,the measured concentration of the endotoxin added to the test solution is within 50-200per cent of the known added endotoxin concentration,after subtraction of any endotoxin detected in the solution without added endotoxin.When the endotoxin recovery is out of the specified ranges,the interfering factors must be removed as described in the section Gel-clot technique,under 1.Preparatory testing,(ii)Test for interfering factors.The efficiency of the treatment is verified by repeating the test for interfering factors.4.TEST(i)ProcedureFollow the procedure described in 3.Preparatory testing,(ii)Test for interfering factors.(ii)CalculationCalculate the endotoxin concentration of each replicate of solution A using the standard curve generated by the series of positive controls,solution C.The test is not valid unless the following 3requirements are met:(a)the result obtained with solution D (negative control)does not exceed the limit of the blank value required in the description of the lysate employed,(b)the results obtained with the series of positive controls,solution C,comply with the requirements for validationdefined under 3.Preparatory testing,(i)Assurance of criteria for the standard curve,(c)the endotoxin recovery,calculated from the endotoxin concentration found in solution B after subtracting the endotoxin concentration found in solution A,is within the range of 50-200per cent.(iii)InterpretationThe preparation being examined complies with the test if the mean endotoxin concentration of the replicates of solution A,after correction for dilution and concentration,is less than the endotoxin limit for the product.5.REAGENTS (i)Lysate solutionDissolve amoebocyte lysate in water for BET or in a buffer,as recommended by the lysate manufacturer,by gentle stirring.Store the reconstituted lysate,refrigerated or frozen,as indicated by the manufacturer.(ii)Amoebocyte lysateAmoebocyte lysate is a lyophilised product obtained from amoebocyte lysate from Horseshoe Crab (Limulus polyphemus or Tachypleus tridentatus ).This reagent refers only to a product manufactured in accordance with the regulations of the competent authority.Amoebocyte lysate reacts with some β-glucans in addition to endotoxins.Amoebocyte lysate preparations which do not react with glucans are available;they are prepared by removing from amoebocyte lysate the G factor,which reacts with glucans,or by inhibiting the G factor reacting system of amoebocyte lysate.These preparations may be used for endotoxin testing in the presence of glucans.(iii)Water for BET (water for bacterial endotoxins test)Water for BET is water for injections R or water produced by other procedures that shows no reaction with the lysate employed at the detection limit of the reagent.The following section is published for information.Test for bacterial endotoxins:guidelines1.INTRODUCTIONEndotoxins from gram-negative bacteria are themost common cause of toxic reactions resulting from contamination of pharmaceutical products with pyrogens;their pyrogenic activity is much higher than that of most other pyrogenic substances.These endotoxins are lipo-polysaccharides.Although there are a small number of pyrogens which possess a different structure,theconclusion is generally justified that the absence of bacterial endotoxins in a product implies the absence of pyrogenic components,provided the presence of non-endotoxin pyrogenic substances can be ruled out.The presence of endotoxins in a product may be masked by factors interfering with the reaction between the endotoxins and the amoebocyte lysate.Hence,the analyst who wishes to replace the rabbit pyrogen test required in a pharmacopoeial monograph by a test for bacterial endotoxins has to demonstrate that a valid test can be carried out on theproduct concerned;this may entail a procedure for removing interfering factors.As indicated in the test for bacterial endotoxins,information must be available on the 2following aspects before a test on a sample can be regarded as valid.1.1.The suitability of the material to be used for the test has to be established.The absence of endotoxins in the water for BET and in the other reagents must be assured and the sensitivity of the amoebocyte lysate must be checked to confirm the sensitivity declared by the manufacturer.1.2.As the product to be examined may interfere with the test,the sensitivity of the amoebocyte lysate is determined in the presence and in the absence of the product under examination.There must be no significant difference between the 2sensitivity values.The test for bacterial endotoxins (2.6.14)indicates methods for removing interfering factors;in the case of interference,another test must be carried out after such a method has been applied to check whether the interference has indeed been neutralised or removed.This annex explains the reasons for the requirements in the test for bacterial endotoxins,then deals with the reading and interpretation of the results.Substitution of the rabbit pyrogen test required in apharmacopoeial monograph by an amoebocyte lysate test constitutes the use of an alternative method of analysis and hence requires validation;some guidance on how to proceed is given in section 11.2.6.14.Bacterial endotoxins EUROPEAN PHARMACOPOEIA5.0The reference method for bacterial endotoxins is stated in the monograph on a given product;where no method is stated,method A is the reference method.If a method other than the reference method is to be used,the analyst must demonstrate that the method is appropriate for this product and gives a result consistent with that obtained with the reference method(see also Section13).2.METHODThe addition of endotoxins to amoebocyte lysate may result in turbidity,precipitation or gelation(gel-clot);only thegel-clot method was used in the Pharmacopoeia as an evaluation criterion in the first type of test for bacterial endotoxins.The advantage was the simplicity of basing the decision to pass or fail the product under examination on the absence or presence of a gel-clot,visible with the naked eye. The quantitative methods described as methods C,D,E and F were developed later:they require more instrumentation, but they are easier to automate for the regular testing of large numbers of samples of the same product. Endotoxins may be adsorbed onto the surface of tubesor pipettes made from certain plastics or types of glass. Interference may appear due to the release of substances from plastic materials.Hence,the materials used should be checked;subsequent batches of tubes or pipettes may have a slightly different composition,and therefore the analyst is advised to repeat such tests on starting with new batchesof materials.The decision to use the test for bacterial endotoxins as a limit test implies first,that a threshold endotoxin concentration must be defined for the product to be tested and second,that the objective of the test is to know whether the endotoxin concentration in the product under examination is below or above this threshold.The quantitative methods C,D,E and F make it possible to determine the endotoxin concentration in the sample under examination,but for compliance with the Pharmacopoeia and in routine quality control the final question is whether or not this concentration exceeds a defined limit.In setting a threshold concentration of endotoxin for the product to be tested,due attention should be paid to the dose of the product:the threshold should be set so as to ensure that as long as the endotoxin concentration in the product remains below this threshold even the maximal dose administered by the intended route per hour does not contain sufficient endotoxin to cause a toxic reaction. When the endotoxin concentration in the product exactly equals the threshold value,gelation will occur,as is the case when the endotoxin concentration is much higher,and the product will fail the test,because the all-or-none character of the test makes it impossible to differentiate between a concentration exactly equal to the threshold concentration and one that is higher.It is only when no gelation occurs that the analyst may conclude that the endotoxin concentration is below the threshold concentration.For products in the solid state,this threshold concentration of endotoxin per mass unit or per International Unit(IU)of product has to be translated into a concentration of endotoxin per millilitre of solution to be tested,as the test can only be carried out on a solution.The case of products that already exist in the liquid state(such as infusion fluids) is discussed below.Endotoxin limit:the endotoxin limit for active substances administered parenterally,defined on the basis of dose,is equal to,where:K=threshold pyrogenic dose of endotoxin perkilogram of body mass in a single hour period,M=maximum recommended dose of product perkilogram of body mass in a single hour period. The endotoxin limit depends on the product and its route of administration and is stated in monographs.Values for K are suggested in Table2.6.14.-5.For other routes,the acceptance criterion for bacterial endotoxins is generally determined on the basis of results obtained during the development of the preparation.Table2.6.14.-5Route of administration K(IU of endotoxin per kilogram ofbody mass per hour) Intravenous 5.0Intravanous,forradiopharmaceuticals2.5Intrathecal0.2Which dilution of the product is to be used in the test to obtain maximal assurance that a negative result means that the endotoxin concentration of the product is less thanthe endotoxin limit and that a positive result means that the lysate detected an endotoxin concentration equal to or greater than the endotoxin limit?This dilution depends on the endotoxin limit and on the sensitivity of the lysate:it is called the Maximum Valid Dilution(MVD)and its value may be calculated as follows:Concentration of test solution:—in mg/ml if the endotoxin limit is specified by mass (IU/mg),—in Units/ml if the endotoxin limit is specified by unit of biological activity(IU/Unit),—in ml/ml if the endotoxin limit is specified by volume (IU/ml).λ=the labelled lysate sensitivity in the gel-clottechnique(IU/ml)or the lowest point usedin the standard curve of the turbidimetric orchromogenic techniques.When the value of the maximum valid dilution is not a whole number,a convenient whole number smaller than the MVD may be used for routine purposes(which means preparing a solution of the product which is less diluted than the MVD indicates).In this case,a negative result indicates that the endotoxin concentration of the product lies below the limit value.However,when the endotoxin concentration of the product in such a test is less than the endotoxin limit but high enough to make the reaction with the lysate resultin a clot,the test may be positive under these conditions. Hence,when a test with this‘convenient’dilution factor is positive,the product should be diluted to the MVD and the test should be repeated.In any case of doubt or dispute the MVD must be used.This stresses the importance of the confirmation of the sensitivity of the lysate.。