ASTM_E_2250_-_02.pdf

Designation:E 2250 02An American National StandardStandard Method forDetermination of Endotoxin Concentration in Water MiscibleMetal Working Fluids1This standard is issued under the fixed designation E 2250;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon(e)indicates an editorial change since the last revision or reapproval.1.Scope1.1 This method covers quantitative methods for the sam-pling and determination of Gram-negative bacterial endotoxinconcentrations in water miscible metalworking fluids(MWF).1.2 Users of this method should be familiar with thehandling of MWF.1.3 This method gives an estimate of the endotoxin concen-tration of the sampled MWF.1.3.1 Used on site,this method gives an indication ofchanges in Gram-negative bacterial contamination in theMWF.1.3.2 This method does not replace Practice E 2144.1.4 This method seeks to minimize inter-laboratory varia-tion but does not ensure uniformity of results.1.5 This method is not intended to relate endotoxin concen-tration in MWF to health effects of inhaled endotoxin.1.6 This standard does not purport to address all of thesafety concerns,if any,associated with its use.It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.Referenced Documents2.1 ASTM Standards:D 1356 Terminology Relating to Sampling and Analysis ofAtmospheres2D 4840 Guide for Sample Chain-of-Custody Procedures3E 1497 Practice for Safe Use of Water-Miscible MetalRemoval Fluids2E 1542 Terminology Relating to Occupational Health andSafety2E 2144 Practice for Personal Sampling and Analysis ofEndotoxin in Metalworking Fluid Aerosols in WorkplaceAtmospheres22.2Government Standard:29 CFR 1910.1450 Occupational Exposure to HazardousChemicals in Laboratories42.3Other Documents:Criteria Document for a Recommended Standard:Occupa-tional Exposure to Metalworking Fluids,1998NIOSH Manual of Analytical Methods(NMAM),4th ed.,Eller and Cassinelli,Eds.,199453.Terminology3.1 For definitions of terms in this method relating to thesubset of metalworking fluids that are used for machining orgrinding to produce the finished part,refer to TerminologiesD 1356 and E 1542.3.2 For definitions pertaining to endotoxins refer to PracticeE 2144.3.3 Definitions of Terms Specific to This Standard:3.3.1 Endotoxin neutralizing protein(ENP),na recombi-nant DNA-produced protein that is labeled with a fluorophoreand is capable of binding endotoxin.3.3.2 Fluorescence Polarization(FP),nan optical tech-nology that relates planar fluorescence to molecular volumeand specific analyte concentration.3.3.2.1 DiscussionIn this context,the molecular volumeof ENP is related to endotoxin concentration.3.3.3 TracerThe term used to indicate fluorophore-labeled ENP.3.3.4 mP,nThe mathematical expression of the planarfluorescence used in fluorescence polarization.3.3.5 endotoxins,na lipopolysaccharide derived from theouter membrane of Gram-negative bacteria.3.3.6 endotoxin unit(EU),na biological potency unitequivalent to the FDA Reference Standard Endotoxin(RSE).Currently,EC-6 is equivalent to 0.1 ng per 1 EU.3.3.7 Limulus amebocyte lysate(LAL)assay,na biologi-cal assay that detects endotoxin.3.3.8 Gram-negative bacteria,nprokaryotic cells thathave a complex cell-wall structure that stain characteristicallywhen subjected to the differential Gram staining procedure.1This method is under the jurisdiction of ASTM Committee E34 on Occupa-tional Health and Safety and is the direct responsibility of Subcommittee E34.50 onHealth and Safety Standards for Metal Working Fluids.Current edition approved October 10,2002.Published December 2002.2Annual Book of ASTM Standards,Vol 11.03.3Annual Book of ASTM Standards,Vol 11.01.4Available from U.S.Government Printing Office Superintendent of Documents,732 N.Capitol St.,NW,Mail Stop:SDE,Washington,DC 20401.5CDC/NIOSH,4676 Columbia Pkwy,Cincinnati,OH 4522619981Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959,United States.4.Summary of Method4.1 A known volume of MWF,typically 1 to 10 L,isdispensed into a 10 3 75 mm borosilicate glass test tubecontaining 1-2 mL of endotoxin-free water.4.2 The diluted MWF,without added tracer,is used todetermine the base fluorescence polarization intensity.4.3 The fluorescent-labeled endotoxin neutralizing protein(tracer)is added to the sample to determine the net planarfluorescence intensity,which is directly related to the endot-oxin concentration.5.Significance and Use5.1 Practice E 2144 uses a biological test(LAL)to measureair-borne endotoxin.The LAL test is not only time consumingand difficult to perform but is also subject to interferences,forexample,biocides,which may render it unreliable to measureMWF endotoxin.5.2 Air-borne end