SIST EN 14031:2003

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Arbeitsplatzatmosphäre - Bestimmung von luftgetragenen EndotoxinenAtmospheres des lieux de travail - Détermination des endotoxines en suspension dans l'airWorkplace atmospheres - Determination of airborne endotoxins13.040.30Kakovost zraka na delovnem mestuWorkplace atmospheresICS:Ta slovenski standard je istoveten z:EN 14031:2003SIST EN 14031:2003en01-marec-2003SIST EN 14031:2003SLOVENSKI
STANDARD



SIST EN 14031:2003



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14031January 2003ICS 13.040.30English versionWorkplace atmospheres - Determination of airborne endotoxinsAtmosphères des lieux de travail - Détermination desendotoxines en suspension dans l'airArbeitsplatzatmosphäre - Bestimmung von luftgetragenenEndotoxinenThis European Standard was approved by CEN on 21 November 2002.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and UnitedKingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2003 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14031:2003 ESIST EN 14031:2003



EN 14031:2003 (E)2ContentspageForeword.3Introduction.41Scope.42Normative references.43Terms and definitions.44Abbreviations.55Overall demands in endotoxin work.65.1General.65.2Demands on operator.65.3Demands on equipment.65.4Demands on glassware.65.5Demands on extraction liquids, water and detergents.65.6Demands on filters.65.7Transportation of samples.65.8Storage of samples at the laboratory.75.9Test for contamination.75.10Sampling documentation.76Extraction.76.1General.76.2Equipment.76.3How to extract.86.4Storage.86.5Extraction documentation.87Analytical methods.87.1General.87.2Equipment.87.3Procedure.97.4Validation.97.5Analytical protocol.98Expression of results.109Precision.1010Test report.10Annex A
(informative)
Properties of endotoxins and sources of exposure.11A.1Identity and physical and chemical properties.11A.2Sources of occupational exposure.11Annex B
(informative)
Principle of LAL-assays.12B.1General.12B.2Detection of inhibition or enhancement of measured endotoxins using spiked samples.12B.3Detection of inhibition or enhancement of measured endotoxins using parallel line assay.12Bibliography.14SIST EN 14031:2003



EN 14031:2003 (E)3ForewordThis document (EN 14031:2003) has been prepared by Technical Committee CEN/TC 137 "Assessment ofworkplace exposure", the secretariat of which is held by DIN.This European Standard shall be given the status of a national standard, either by publication of an identical text orby endorsement, at the latest by July 2003, and conflicting national standards shall be withdrawn at the latest byJuly 2003.Annexes A and B are informative.According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain,Sweden, Switzerland and the United Kingdom.SIST EN 14031:2003



EN 14031:2003 (E)4IntroductionEndotoxins are integral components of the outer membrane of gramnegative bacteria which are composed ofproteins, lipids, and lipopolysaccharides. The term 'endotoxin' refers to the toxin as present on the bacterial cellwall. Lipopolysaccharides of gram negative bacteria refer to a class of pure lipid carbohydrate molecules (free ofprotein and other cell wall components) that are held responsible for most of the biological properties characteristicof bacterial endotoxins. In annex A a brief overview is given with respect to physical and chemical properties ofendotoxins and sources of exposure in the occupational environment.Endotoxins are believed to play an important role in the development of organic dust related diseases in exposedworkers.It is important to assess occupational exposure to airborne endotoxins in a representative way to evaluate theexposure. There are, however, at present no generally accepted procedures for the measurement of environmentalendotoxins, thus different practices continue to exist. Rigorous standardization with respect to sampling media,extraction media, analytical methods and storage conditions for endotoxins are needed to obtain results that arecomparable between studies. By adhering to the recommendations outlined in this standard for choice of sampling,storage of samples, extraction and analytical procedures uncertainties in exposure assessment can be reducedand controlled, allowing comparable and representative measurements to be made.1 ScopeThis European Standard provides guidelines for the assessment of workplace exposure to airborne bacterialendotoxins. The standard provides methods for sampling, transportation, and storage of samples anddetermination of endotoxins.2 Normative referencesThis European Standard incorporates by dated or undated reference, provisions from other publications. Thesenormative references are cited at the appropriate place in the text, and the publications are listed hereafter. For datedreferences, subsequent amendments to or revisions of any of these publications apply to this European Standard onlywhen incorporated in it by amendment or revision. For undated references, the latest edition of the publicationreferred to applies (including amendments).EN 481:1993, Workplace atmospheres – Size fraction definitions for measurement of airborne particles.EN 1232, Workplace atmospheres – Pumps for personal sampling of chemical agents – Requirements and testmethods.EN 12919, Workplace atmospheres – Pumps for the sampling of chemical agents with a volume flow rate of over5 l/min - Requirements and test methods.3 Terms and definitionsFor the purposes of this European Standard, the following terms and definitions apply.3.1bioaerosolairborne particles with biological originNOTESee EN 13098:2000, 3.3, notes.3.2control standard endotoxinstandard that is traceable to the RSESIST EN 14031:2003



EN 14031:2003 (E)53.3endotoxinsee EN 13098:2000.3.4endotoxinfree liquidswater and solutions with <0,2 EU/mlNOTEEndotoxinfree and pyrogenfree are used interchangeable in the literature.3.5endotoxin unitsee EN 13098:2000NOTEEndotoxin concentrations in samples are referenced to a defined reference standard endotoxin (RSE) andsubsequently expressed in units based on the activity of the reference material in a LAL assay.3.6exposure (by inhalation)situation in which a chemical or biological agent is present in air which is inhaled by a person[EN 1540:1998]3.7inhalable particle size fractioninhalable fraction which constitutes the mass fraction of total airborne particles which is inhaled through the noseand mouth[EN 481:1993]3.8LAL-assayfunctional assay to measure endotoxin concentrationsNOTEThe method is highly sensitive and based on the activation of a clotting enzyme present in the lysate of hemolymph ofthe Limulus polyphemus (Horseshoe crab).3.9lipopolysaccharidesLPS present in gramnegative bacteria are water-soluble and stable molecules composed of lipid andpolysaccharideNOTEThe lipid part of LPS is termed 'lipid A' and is essential for the toxic properties of LPS. The terms 'endotoxins' and'lipopolysaccharides' are often used interchangeably in the scientific literature.3.10reference standard endotoxinpurified LPS from Escherichia coli that serves as an international reference standard4 AbbreviationsCSEcontrol standard endotoxinEU endotoxin unitsLAL limulus amoebocyte lysateLPS lipopolysaccharidesSIST EN 14031:2003



EN 14031:2003 (E)6RSEreference standard endotoxin5 Overall demands in endotoxin work5.1 GeneralEnvironmental monitoring of endotoxins is performed by sampling inhalable aerosols. Aerosols are sampled on afilter using a pump to draw air through the filter. For assessing occupational exposure personal sampling shall beused for example when comparing with exposure guidelines. Area sampling can be used to identify sources ofexposure.5.2 Demands on operatorThe operator that performs the sampling shall be trained in aseptic work conditions, have knowledge of samplingequipment and know how to carry out the sampling. The operator shall avoid contamination of the sample during allphases of sampling.5.3 Demands on equipmentThe pumps shall fulfil the requirements of the standards for pumps for personal and static sampling (EN 1232 andEN 12919). Personal samplers shall collect the inhalable aerosol fraction as defined by EN 481.The filter holder or cassette should be rendered endotoxinfree. Prior to sampling, reusable samplers should becleaned thoroughly so that contamination of the samples are avoided. Cleaning agents shall be removed bysubsequent washing in endotoxinfree water. Disposable samplers do not need to be sterilised when one canassume the manufacturing process to be fairly endotoxinfree. Possible contamination shall be controlled by usingblank filters (see 5.9).5.4 Demands on glasswareAll other materials used during sampling, transportation and storage shall be rendered endotoxinfree. Glasswarecan be rendered endotoxinfree by heating at least at 180 °C for 4 h.5.5 Demands on extraction liquids, water and detergentsAll liquids used shall be endotoxinfree.5.6 Demands on filtersBinderfree glass fibre filters should be used for endotoxin sampling while these filters usually give the highestrecoveries. Other types of filters can be used. If any other filter is used it shall be validated toward filters of glassfibre.NOTESeveral types of filters (cellulose esters, polyvinylchloride, polytetrafluoroethylene and polycarbonate) are commonlyused but some material adsorb endotoxins and thereby give false low values.5.7 Transportation of samplesAfter sampling filters shall be prepared for transport by sealing in a container or sampler cassette. The sample shallbe transported in dry conditions, preferably with a dehumidifier. Samples can be transported at room- or outsidetemperature but conditions for transport shall be documented and include transportation time, temperature etc. Iftransportation time is over 24 h the samples shall be transported in a condition that prevents the filters from gettingmoist during transportation, i.e. with a dehumidifier or frozen.SIST EN 14031:2003



EN 14031:2003 (E)75.8 Storage of samples at the laboratorySamples shall never be repeatedly frozen and thawed, as this may effect the detectable endotoxin content of thesample. Endotoxins are very stable when frozen. If the sample is not extracted within a few days after arrival to thelaboratory the sample shall be stored at approximately –20 °C or below.5.9 Test for contaminationTo test for contamination a series of at least 2 blank filters shall be included in each sampling session. Except forthe actual sampling, blank filters shall be treated equal to the other filters that are used for sampling.5.10 Sampling documentationA list of sampling operations shall be documented to obtain comparable and reliable concentration values ofendotoxins. The sampling document
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