1、Designation: D 7102 04Standard Guide forDetermination of Endotoxin on Sterile Medical Gloves1This standard is issued under the fixed designation D 7102; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A n
2、umber in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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 g
3、loves. 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
4、formation, 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 co
5、vers 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 sterilegloves.1.3 This guide is not applicable to the determ
6、ination ofpyrogens other than bacterial endotoxins.1.4 The sample preparation method described must be usedregardless of the test method selected. This method does notdescribe laboratory test method validation, analyst qualifica-tion, or reagent confirmation. Product-specific validation isaddressed.
7、1.5 The safe and proper use of medical gloves is beyond thescope of this guide.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 dete
8、rmine the applica-bility of regulatory requirements prior to use.2. Referenced Documents2.1 EN Standard:2EN 455-3:1999 Medical Gloves for Single UsePart 3:Requirements and Testing for Biological Evaluation2.2 ANSI Standard:2ANSI/AAMI ST 72:2002 Bacterial EndotoxinsTestMethodologies, Routine Monitori
9、ng and Alternatives toBatch Testing3. Terminology3.1 Definitions:3.1.1 bacterial endotoxin test (BET)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-
10、tion.3.1.2 batchdefined quantity of intermediate or finishedproduct produced in a defined cycle of manufacture that is saidto 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 propo
11、rtional to the interac-tion of LAL and endotoxin.3.1.4 control standard endotoxin (CSE)purified endotoxinproduct supplied at 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
12、 in pairs anda single glove when packaged singly.3.1.6 endotoxinhigh molecular weight, heat stable com-plex associated with the cell wall of gram-negative bacteriathat is pyrogenic in humans and specifically interacts with LAL.1This guide is under the jurisdiction of ASTM Committee D11 on Rubber and
13、is the direct responsibility of Subcommittee D11.40 on Consumer Rubber Products.Current edition approved Dec. 1, 2004. Published January 2005.2Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036.1Copyright ASTM International, 100 Barr Harbor Driv
14、e, PO Box C700, West Conshohocken, PA 19428-2959, United States.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 material).3.1.7.1 DiscussionThe FDAs end
15、otoxin 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.3.1.9 enhancementa type of interference th
16、at 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 onthe proportional reaction of endotoxin with LAL.3.1.11 inhibitionBET anomaly wherein a non-endotoxinsubstance
17、, usually contributed by the sample, elicits a testreaction less than the amount of endotoxin actually present.3.1.12 inhibition/enhancement testtest used to determinewhether a particular BET sample contains factors that diminishits accuracy of the BET either by enhancement or inhibition ofthe resul
18、ts.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 polyphemus or Tachypleus tridentatus(TAL), which forms a clot when brought into cont
19、act withsubstances containing endotoxin.3.1.15 lotsee batch.3.1.16 lipopolysaccharide (LPS)the gram-negative cellwall component typically composed of lipid A, a core polysac-charide, and an O-side chain sugar moiety.3.1.17 LAL reagent water (LRW)LAL reagent water thathas been validated to contain no
20、 detectable endotoxin.3.1.18 maximum valid dilution (MVD)the highest dilutiona sample is permitted to attain in diluting out interferingsubstances 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 l
21、abel medical devices that containendotoxin below a specified level.3.1.20 pyrogenany substance that can induce a fever.Endotoxins are one type of pyrogen.3.1.21 pyrogenica term used to describe healthcare prod-ucts with endotoxin levels above specified limits.3.1.22 reference standard endotoxin (RSE
22、)the USPendot-oxin reference standard defined to have a potency of 10 000USP EUs per vial.3.1.23 turbidimetric techniqueBET methodology that de-tects or quantifies endotoxin based on the level of turbiditycreated proportional to the interaction of LAL and endotoxin.4. Summary of Guide4.1 A standard
23、method of sample preparation is specified inthis guide.4.2 Four variations of endotoxin determination test methodsare identified and briefly described to facilitate selection of theappropriate method. The reader is referred to the referencedstandards for complete instructions.5. Significance and Use
24、5.1 This guide establishes a standard sample preparationmethod and provides a description of four 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 appropriat
25、e 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 paramet
26、ers, 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 Prepar
27、ation, and ExtractionNOTE 1All gloves must follow this sampling plan, sample prepara-tion, 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 the
28、 batch size. Three percentof the batch should be tested with a minimum of 3 pairs ofgloves and a maximum of 10 pairs of gloves per batch. Forbatch sizes under 30 units, two pairs of gloves may beanalyzed.6.1.1 Samples selected for testing should be produced andselected in the finished form. This inc
29、ludes all factors thatmight affect or contribute to the levels of endotoxin (forexample, post-packaging and sterilization).6.2 Sample Extraction:6.2.1 Handle everything with pyrogen-free instruments.Perform all extractions in non-pyrogenic containers.6.2.2 A sample extraction is prepared by immersin
30、g theoutside surface of the gloves in LRW. The extractions should beperformed with 40 mL of LRW per pair of gloves, for 40 to 60min at a temperature between 37 to 40C.6.2.3 The extraction should be performed in a way to ensurethat all surfaces of the gloves that would have patient contact,come in co
31、ntact with the extraction medium. For example, theglove may be lowered into a flask containing 40 mL of LRWwith the exterior of the glove in contact with the water and thecuff (#2 cm) folded over the opening at the neck of the flask.6.2.4 After the sample has been extracted, remove the glove.If appr
32、opriate, the second glove of the pair can then beextracted in the same flask with the extraction fluid of the firstglove or both gloves may be extracted in the 40 mL LRW at thesame time by both being suspended simultaneously in the flask,held in place by a non-pyrogenic stopper by the cuff at theope
33、ning of the 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 clarification or the removal of particulatesD7102042are acceptable treatment methods a
34、s 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 frozen or refrigerated to prevent microbial growth,which will increase endotoxin levels.7. Test Methods7.1
35、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 and assessment of the product and testingfacility. Current
36、 techniques are: (a) Gel Clot, (b) KineticTurbidimetric, (c) Kinetic Chromogenic (Colorimetric), and(d) Endpoint Chromogenic. These may be found in the FDAguidelines (1-3)3, U.S. Pharmacopoeias (4-7), ANSI/AAMI ST72, and EN 455-3.7.1.1 Gel Clot TechniquesThe gel clot methods are thesimplest of the B
37、ET methods, both in terms of technicalexpertise required to perform a valid assay and datainterpretation/analysis. Investment in equipment is minimal,requiring only a properly qualified and maintained waterbath orheating block and accessories. In the gel-clot test, equalvolumes of test sample dilute
38、d to a validated concentration andLAL reagent are mixed in a 10 by 75-mm glass test tube. Afterincubation, individual 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
39、than a firm gel is scored as a negative test.The disadvantage to this test is that it is a qualitative testmethod and 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 o
40、f EU in the sample can then be determined by incorpo-rating the dilution factor into the calculation.7.1.2 Kinetic Turbidimetric TechniqueThe kinetic methodfor the turbidimetric technique measures the amount of time ittakes for a series of standards to reach a pre-determined opticaldensity (OD), som
41、etimes called the onset OD or reaction OD.A standard curve is constructed by plotting the log of the onsetor reaction time (that is, the time it takes for each standard orsample to reach the onset OD) as a function of the log of theendotoxin concentration. This log/log treatment of the dataresults i
42、n a linear standard curve. The range of the curve for akinetic assay is up to four logs as compared to the one logcurve generated in the gel-clot method. The resulting curve isconstructed using linear regression analysis across the ob-served points. A correlation coefficient of r=0.980 is theminimum
43、 linearity requirement for a valid kinetic method. Theendotoxin content of the unknown sample is calculated byinterpolation from the standard curve using the logarithm of theonset time of the sample. The kinetic methods may beperformed in microtiterplates, glass tubes, or other validatedtechnology.
44、Kinetic methods require qualified equipment toread the results and software with a statistical package (regres-sion analysis) for the construction of standard curves andanalysis of samples. The disadvantage to the kinetic turbidi-metric technique is that it is not appropriate for turbid samples.7.1.
45、3 Kinetic Chromogenic TechniqueThe kinetic meth-ods for the chromogenic technique measures the amount oftime it takes for a series of standards to reach a pre-determinedcolor intensity, sometimes called the onset OD or reaction OD.The technique is the same as described under the kineticturbidimetric
46、 technique. The disadvantage to the chromogenictest method is that it has often been found to be more subjectto interference, in comparison with the kinetic turbidimetricmethod. Laboratories may choose a modification of thistechnique identified as Endpoint Chromogenic.7.1.4 Endpoint Chromogenic Tech
47、niqueThis method is amodification of the kinetic chromogenic assay. Rather thanreading continuous OD measurements, the color intensity ismeasured at the end of a set time period. The color intensity iscompared with the control series to give a quantitative endot-oxin result. The disadvantage to the
48、chromogenic test methodis that it has often been found to be more subject to interfer-ence, in comparison with the turbidimetric methods.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.
49、6 For samples that cannot be tested by any of the BETmethods because of non-removable inhibition or enhancement,the Rabbit Pyrogen Test may be utilized.7.1.6.1 The Pyrogen Test is an in vivo test method. Threerabbits are injected intravenously with the pooled extracts of 10test samples at a dose not to exceed 10 mL/kg within a periodof not more than 10 min. Perform the injection after warmingthe sample extracts to 37 6 2C. Record animal temperaturesat 30-min intervals between 1 and 3 h subsequent to injection.The complete details of test animal, facil
