1、Designation: D7102 17Standard Guide forDetermination of Endotoxin on Sterile Medical Gloves1This standard is issued under the fixed designation D7102; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A num
2、ber in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.INTRODUCTIONThis guide is established and designed to determine the qualitative or quantitative presence ofbacterial endotoxin on sterile medical glov
3、es. Bacterial endotoxins are found in the outer membraneof Gram negative bacteria and may contaminate gloves during the manufacturing process. Conse-quences of endotoxin introduced into a patient during invasive procedures are dose dependent and mayinclude inflammation, fever, nausea, pain, clot for
4、mation, hypoglycemia and reduced profusion of theheart, kidney, and liver as well as endotoxic shock. Endotoxins are not inactivated by routine methodsutilized in the routine sterilization of medical gloves including irradiation (gamma or E-beam),ethylene oxide, or steam.1. Scope1.1 This guide cover
5、s a selection of methodologies for thedetermination of bacterial endotoxin on gloves when such adetermination is appropriate.1.2 As bacteria may continue to grow on non-sterile gloves,reportable endotoxin levels are only appropriate for gloveslabeled as sterile. Because most environments containendo
6、toxin, once a box of gloves is opened and the gloves aremanipulated, endotoxin levels will increase making it inappro-priate to report endotoxin levels on boxed gloves (ex. exami-nation gloves). This is true even if the box had undergonesterilization prior to distribution.1.3 This guide may also be
7、appropriate for internal qualitycontrol or alert purposes at different stages of manufacturing orduring process change evaluations.1.4 This guide is not applicable to the determination ofpyrogens other than bacterial endotoxins.1.5 The sample preparation method described must be usedregardless of th
8、e test method selected. This method does notdescribe laboratory test method validation, analystqualification, or reagent confirmation. Product-specific valida-tion is addressed.1.6 The safe and proper use of medical gloves is beyond thescope of this guide.1.7 This standard does not purport to addres
9、s 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 requirements prior to use.1.8 This international standard was developed in accor-dan
10、ce with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 EN Stand
11、ard:2EN 455-3:2015 Medical Gloves for Single UsePart 3:Requirements and Testing for Biological Evaluation2.2 ANSI Standard:2ANSI/AAMI ST 72:2011 Bacterial EndotoxinsTestMethodologies, Routine Monitoring and Alternatives toBatch Testing3. Terminology3.1 Definitions:3.1.1 bacterial endotoxin test (BET
12、)a method for deter-mining the qualitative or quantitative presence of endotoxin inan aqueous test sample utilizing Limulus amebocyte lysate(LAL) reagent and measuring the resulting proportional reac-tion.1This guide is under the jurisdiction of ASTM Committee D11 on Rubber andRubber-like Materials
13、and is the direct responsibility of Subcommittee D11.40 onConsumer Rubber Products.Current edition approved May 1, 2017. Published June 2017. Originallyapproved in 2004. Last previous edition approved in 2010 as D7102 10. DOI:10.1520/D7102-17.2Available from American National Standards Institute (AN
14、SI), 25 W. 43rd St.,4th Floor, New York, NY 10036.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the
15、Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.13.1.2 batchdefined quantity of intermediate or finishedproduct produced in a defined cycle of manufacture that is saidt
16、o be of uniform quality.3.1.3 chromogenic (colorimetric) techniqueBET method-ology that quantifies or detects endotoxin on the basis of ameasured color-producing reaction proportional to the interac-tion of LAL and endotoxin.3.1.4 control standard endotoxin (CSE)purified endotoxinproduct supplied at
17、 a known potency and utilized as a standardcontrol in endotoxin testing.3.1.5 devicewith regard to medical gloves, a device isdefined as a pair of gloves when they are packaged in pairs anda single glove when packaged singly.3.1.6 endotoxinhigh molecular weight, heat stable com-plex associated with
18、the cell wall of gram-negative bacteriathat is pyrogenic in humans and specifically interacts with LAL.3.1.7 endotoxin unit (EU)the standard unit of measure forendotoxin activity initially established relative to the activity in0.2 ng of the U.S. Reference Standard Endotoxin (USPstandard reference m
19、aterial).3.1.7.1 DiscussionThe FDAs endotoxin standard and thatof the World Health Organizations International EndotoxinStandard (IU) are sub lots of the same endotoxin preparation,making EU and IU equal.3.1.8 endpoint (gel clot)last positive (coagulated or gelclot) tube in a series of dilutions.3.1
20、.9 enhancementa type of interference that renders testresults with higher values than the amount of endotoxinpresent.3.1.10 gel-clot techniqueBET methodology that can beused to detect or quantify the presence of endotoxin based onclotting of the lysate reagent (gel formation) in the presence ofendot
21、oxin.3.1.11 inhibitionBET anomaly wherein a non-endotoxinsubstance, usually contributed by the sample, elicits a testreaction less than the amount of endotoxin actually present.3.1.12 inhibition/enhancement (suitability) testtest usedto determine whether a particular BET sample contains factorsthat
22、diminish its accuracy of the BET either by enhancement orinhibition of the results.3.1.13 interfering substancesthose substances that causeinhibition or enhancement.3.1.14 Limulus amebocyte lysate (LAL)the reagent ex-tracted from amebocytes in the circulatory system of thehorseshoe crab Limulus poly
23、phemus or Tachypleus tridentatus(TAL), which forms a clot when brought into contact withsubstances containing endotoxin.3.1.15 lotsee batch.3.1.16 lipopolysaccharide (LPS)the gram-negative cellwall component typically composed of lipid A, a corepolysaccharide, and an O-side chain sugar moiety.3.1.17
24、 LAL reagent water (LRW)LAL reagent water thathas been validated to contain no detectable endotoxin, typicallyavailable from the lysate manufacturer for automated testsystems.3.1.18 maximum valid dilution (MVD)the highest dilutiona sample is permitted to attain in diluting out interferingsubstances
25、while still being capable of detecting endotoxin inthe sample extract.3.1.19 non-pyrogenicdescribes a product that does notinduce a fever. Also used to label medical devices that containendotoxin below a specified level.3.1.20 pyrogenany substance that can induce a fever.Endotoxins are one type of p
26、yrogen.3.1.21 pyrogenica term used to describe healthcare prod-ucts with endotoxin levels above specified limits.3.1.22 reference standard endotoxin (RSE)the USP endo-toxin reference standard defined to have a potency of 10 000USP EUs per vial.3.1.23 turbidimetric techniqueBET methodology that de-te
27、cts or quantifies endotoxin based on the level of turbiditycreated proportional to the interaction of LAL and endotoxin.3.1.24 water for BETwater that shows no reaction withthe lysate employed, at the detection limit of the reagent.Examples LRW and Water of Injection.4. Summary of Guide4.1 A standar
28、d method of sample preparation is specified inthis guide.4.2 Three variations of endotoxin determination test meth-ods are identified and briefly described to facilitate selection ofthe appropriate method. The reader is referred to the referencedstandards for complete instructions.5. Significance an
29、d Use5.1 This guide establishes a standard sample preparationmethod and provides a description of three established andrecognized test methods for the determination of endotoxin onmedical gloves. If interferences in a sample yield suspectresults, a second method should be used.5.2 This guide is appr
30、opriate for testing final product thathas been subjected to all processes that could influence the finalendotoxin level (either microbial contamination or processingagents/raw materials contaminated with endotoxin). As rawmaterials and processing conditions vary from lot to lot withregard to these p
31、arameters, it is appropriate to test for endo-toxin on a routine basis if a product endotoxin claim is to bemade (for example, non-pyrogenic). The user may find itbeneficial to incorporate endotoxin testing for vulnerable areasof their manufacturing process as an alert mechanism.6. Sampling, Sample
32、Preparation, and ExtractionNOTE 1All gloves must follow this sampling plan, samplepreparation, and extraction method regardless of assay method chosen.6.1 SamplingThe bacterial endotoxin test shall be carriedout for each batch of gloves where a limit has been set. Thesampling plan should be based on
33、 the batch size. Refer to Table1.6.1.1 Samples selected for testing should be produced andselected in the finished form. This includes all factors thatD7102 172might contribute to the levels of endotoxin (for example, anymanufacturing, physical handling, packaging, or delay insterilization).6.2 Samp
34、le Extraction:6.2.1 Handle everything with pyrogen-free instruments.Perform all extractions in non-pyrogenic containers.6.2.1.1 Glassware or Other Heat-Stable MaterialsCommonly used minimum time and temperature of 30 min at250C in hot air oven using validated process may be applied.Refer to U.S. Pha
35、rmacopoeia (1).36.2.1.2 PlasticsUse materials that have been shown to befree of detectable endotoxin and do not interfere in the test.6.2.2 A sample extraction is prepared by immersing theoutside surface of the gloves in Water for BET. The standardextraction method is to soak or immerse a sample or
36、flush thefluid pathway with extracting fluid that has been heated to 376 1.0C, keeping the extracting fluid in contact with therelevant surface(s) for no less than 1 h. Alternate extraction orrinsing methods may be used, but must be demonstrated to beequivalent or better than the standard method.6.2
37、.3 The extraction should be performed in a way to ensurethat all surfaces of the gloves that would have patient contact,come in contact with the extraction medium. For example, theglove may be lowered into a flask containing sufficient volumeof Water for BET for the full exterior of the glove to be
38、incomplete contact with the water and the cuff (2 cm) foldedover the opening at the neck of the flask and held in place bya non-pyrogenic stopper by the cuff.6.2.4 Extracts or samples may be pooled for testing. Forexample, a glove may be extracted, removed and a secondglove may be suspended in the s
39、ame extraction liquid.Alternately, both gloves may be extracted simultaneously inthe same flask.6.2.5 Powder and other particulate matter can interfere withendotoxin determination assays. Interference should be over-come with sample dilution. Neither extract filtration norcentrifugation for clarific
40、ation or the removal of particulatesare acceptable treatment methods as endotoxin can be coinci-dentally removed from the test sample.6.2.6 Test the sample by one of the methods identified inSection 7, Test Methods. If not tested immediately, the sampleshould be refrigerated to prevent microbial gro
41、wth, which mayincrease endotoxin levels.7. Test Methods7.1 Bacterial Endotoxin Test (BET) MethodsThe testinglaboratory may choose bacterial endotoxin testing techniques,described in the various compendia, guidelines, and productinserts. The choice of the technique should be made aftercareful thought
42、 and assessment of the product and testingfacility. Current techniques are: (a) Gel Clot, (b) Turbidimetric,and (c) Chromogenic (Colorimetric). These may be found inFDA Guidelines (2), U.S. Pharmacopoeia (3-5), ANSI/AAMIST 72, and EN 455-3/European Pharrmacopeia (6). Thoroughunderstanding of the lis
43、ted standards is required. For subjectsnot covered in compendial procedures refer to FDA guidance(2).7.1.1 Gel Clot TechniquesIn the gel-clot test, equal vol-umes of test sample diluted to a validated concentration andLAL reagent are mixed in a 10 by 75-mm glass test tube. Afterincubation, individua
44、l test tubes are carefully removed fromthe incubating device and slowly inverted 180. A firm gel thatmaintains its integrity upon inversion is scored as a positivetest. Anything other than a firm gel is scored as a negative test.The disadvantage to this test is that it is a qualitative testmethod an
45、d has a lower test sensitivity. The test may be madesemi-quantitative by diluting positive test samples and assay-ing each dilution until an end point (no clot) is obtained. Thelevel of EU in the sample can then be determined by incorpo-rating the dilution factor into the calculation.7.1.2 Turbidime
46、tric TechniqueThis technique measuresthe increases in reactant turbidity by either assaying thequantitative relationship between endotoxin concentration andturbidity (determined by optical density readings) of thereaction mixture at the end of a pre-determined incubation time(endpoint variant) or by
47、 measuring the time required, alsorefered to as onset time, for the reaction to produce apredetermined turbidity level (kinetic variant). The disadvan-tage to the kinetic turbidimetric technique is that it is notappropriate for turbid samples.7.1.3 Chromogenic TechniqueThis assay measures thechromop
48、hore release from a suitable chromogenic peptide bythe reaction of endotoxin with the lysate. This method also haskinetic and a chromogenic variants as described in 7.1.2,however, the detectable change is color intensity instead ofturbidity. The disadvantage to the chromogenic test method isthat it
49、has often been found to be more subject to interference,in comparison with the kinetic turbidimetric method.7.1.4 For details regarding the performance and test param-eters for the gel clot, turbidimetric, and chromogenic testmethods refer to U.S. Pharmacopoeia (3) and ANSI/AAMI ST72.7.1.5 A batch of gloves that fails one of the BET methodsdescribed above may be retested once by the same method usedoriginally or by one of the other methods.7.1.6 For samples that cannot be tested by any of the BETmethods because of non-removable inhi
