1、Designation: D7102 10D7102 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 revisio
2、n. A number 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 medi
3、cal gloves. Bacterial endotoxins are found in the outer membraneof gramGram negative bacteria and may contaminate gloves during the manufacturing process.Consequences of endotoxin introduced into a patient during invasive procedures are dose dependentand may include inflammation, fever, nausea, pain
4、, clot formation, hypoglycemia and reducedprofusion of the heart, kidney, and liver as well as endotoxic shock. Endotoxins are not inactivated byroutine methods utilized in the routine sterilization of medical gloves including irradiation (gamma orE-beam), ethylene oxide, or steam.1. Scope1.1 This g
5、uide covers a selection of methodologies for the determination 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 gloves labeledas sterile. Because most environments
6、 contain endotoxin, once a box of gloves is opened and the gloves are manipulated, endotoxinlevels will increase making it inappropriate to report endotoxin levels on boxed gloves (ex. examination gloves). This is true evenif the box had undergone sterilization prior to distribution.1.3 This guide m
7、ay also be appropriate for internal quality control or alert purposes at different stages of manufacturing orduring process change evaluations.1.4 This guide is not applicable to the determination of pyrogens other than bacterial endotoxins.1.5 The sample preparation method described must be used re
8、gardless of the test method selected. This method does notdescribe laboratory test method validation, analyst qualification, or reagent confirmation. Product-specific validation is addressed.1.6 The safe and proper use of medical gloves is beyond the scope of this guide.1.7 This standard does not pu
9、rport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatoryrequirements prior to use.1.8 This international standard was developed
10、 in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.
11、1 EN Standard:2EN 455-3:1999455-3:2015 Medical Gloves for Single UsePart 3: Requirements and Testing for Biological Evaluation2.2 ANSI Standard:2ANSI/AAMI ST 72:200272:2011 Bacterial EndotoxinsTest Methodologies, Routine Monitoring and Alternatives to BatchTesting1 This guide is under the jurisdicti
12、on ofASTM Committee D11 on Rubber and Rubber-like Materials and is the direct responsibility of Subcommittee D11.40 on ConsumerRubber Products.Current edition approved May 1, 2010May 1, 2017. Published June 2010June 2017. Originally approved in 2004. Last previous edition approved in 20042010 asD710
13、2 04.D7102 10. DOI: 10.1520/D7102-10.10.1520/D7102-17.2 Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have bee
14、n made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document
15、.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13. Terminology3.1 Definitions:3.1.1 bacterial endotoxin test (BET)a method for determining the qualitative or quantitative presence of endotoxin in anaqueous test sample utilizing Limul
16、us amebocyte lysate (LAL) reagent and measuring the resulting proportional reaction.3.1.2 batchdefined quantity of intermediate or finished product produced in a defined cycle of manufacture that is said to beof uniform quality.3.1.3 chromogenic (colorimetric) techniqueBET methodology that quantifie
17、s or detects endotoxin on the basis of a measuredcolor-producing reaction proportional to the interaction of LAL and endotoxin.3.1.4 control standard endotoxin (CSE)purified endotoxin product supplied at a known potency and utilized as a standardcontrol in endotoxin testing.3.1.5 devicewith regard t
18、o medical gloves, a device is defined as a pair of gloves when they are packaged in pairs and a singleglove when packaged singly.3.1.6 endotoxinhigh molecular weight, heat stable complex associated with the cell wall of gram-negative bacteria that ispyrogenic in humans and specifically interacts wit
19、h LAL.3.1.7 endotoxin unit (EU)the standard unit of measure for endotoxin activity initially established relative to the activity in 0.2ng of the U.S. Reference Standard Endotoxin (USP standard reference material).3.1.7.1 DiscussionThe FDAs endotoxin standard and that of the World Health Organizatio
20、ns International Endotoxin Standard (IU) are sub lots ofthe same endotoxin preparation, making EU and IU equal.3.1.8 endpoint (gel clot)last positive (coagulated or gel clot) tube in a series of dilutions.3.1.9 enhancementa type of interference that renders test results with higher values than the a
21、mount of endotoxin present.3.1.10 gel-clot techniqueBET methodology that can be used to detect or quantify the presence of endotoxin based on theproportional reaction of endotoxin with clotting of the lysate reagent (gel formation) in the presence of endotoxin.LAL.3.1.11 inhibitionBET anomaly wherei
22、n a non-endotoxin substance, usually contributed by the sample, elicits a test reactionless than the amount of endotoxin actually present.3.1.12 inhibition/enhancement (suitability) testtest used to determine whether a particular BET sample contains factors thatdiminish its accuracy of the BET eithe
23、r by enhancement or inhibition of the results.3.1.13 interfering substancesthose substances that cause inhibition or enhancement.3.1.14 Limulus amebocyte lysate (LAL)the reagent extracted from amebocytes in the circulatory system of the horseshoe crabLimulus polyphemus or Tachypleus tridentatus (TAL
24、), which forms a clot when brought into contact with substances containingendotoxin.3.1.15 lotsee batch.3.1.16 lipopolysaccharide (LPS)the gram-negative cell wall component typically composed of lipidA, a core polysaccharide,and an O-side chain sugar moiety.3.1.17 LAL reagent water (LRW)LAL reagent
25、water that has been validated to contain no detectable endotoxin.endotoxin,typically available from the lysate manufacturer for automated test systems.3.1.18 maximum valid dilution (MVD)the highest dilution a sample is permitted to attain in diluting out interfering substanceswhile still being capab
26、le of detecting endotoxin in the sample extract.3.1.19 non-pyrogenicdescribes a product that does not induce 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 pyrogen.3.1.21 pyroge
27、nica term used to describe healthcare products with endotoxin levels above specified limits.3.1.22 reference standard endotoxin (RSE)the USP endotoxin reference standard defined to have a potency of 10 000 USPEUs per vial.3.1.23 turbidimetric techniqueBET methodology that detects or quantifies endot
28、oxin based on the level of turbidity createdproportional to the interaction of LAL and endotoxin.3.1.24 water for BETwater that shows no reaction with the lysate employed, at the detection limit of the reagent. Examples LRW and Water of Injection.D7102 1724. Summary of Guide4.1 A standard method of
29、sample preparation is specified in this guide.4.2 FourThree variations of endotoxin determination test methods are identified and briefly described to facilitate selection ofthe appropriate method. The reader is referred to the referenced standards for complete instructions.5. Significance and Use5.
30、1 This guide establishes a standard sample preparation method and provides a description of fourthree established andrecognized test methods for the determination of endotoxin on medical gloves. If interferences in a sample yield suspect results,a second method should be used.5.2 This guide is appro
31、priate for testing final product that has been subjected to all processes that could influence the finalendotoxin level (either microbial contamination or processing agents/raw materials contaminated with endotoxin). As rawmaterials and processing conditions vary from lot to lot with regard to these
32、 parameters, it is appropriate to test forendo-toxinendotoxin on a routine basis if a product endotoxin claim is to be made (for example, non-pyrogenic). The user mayfind it beneficial to incorporate endotoxin testing for vulnerable areas of their manufacturing process as an alert mechanism.6. Sampl
33、ing, Sample Preparation, and ExtractionNOTE 1All gloves must follow this sampling plan, sample preparation, and extraction method regardless of assay method chosen.6.1 SamplingThe bacterial endotoxin test shall be carried out for each batch of gloves where a limit has been set.The samplingplan shoul
34、d be based on the batch size. Three percentRefer to Table 1of the batch should be tested with a minimum of 3 pairs ofgloves and a maximum of 10 pairs of gloves per batch. For batch sizes under 30 units, two pairs of gloves may be analyzed6.1.1 Samples selected for testing should be produced and sele
35、cted in the finished form. This includes all factors that mightcontribute to the levels of endotoxin (for example, any manufacturing, physical handling, packaging, or delay in sterilization).6.2 Sample Extraction:6.2.1 Handle everything with pyrogen-free instruments. Perform all extractions in non-p
36、yrogenic containers.6.2.1.1 Glassware or Other Heat-Stable MaterialsCommonly used minimum time and temperature of 30 min at 250C in hotair oven using validated process may be applied. Refer to U.S. Pharmacopoeia (1).36.2.1.2 PlasticsUse materials that have been shown to be free of detectable endotox
37、in and do not interfere in the test.6.2.2 A sample extraction is prepared by immersing the outside surface of the gloves in LRW.Water for BET. The extractionsshould be performed with 40 mL ofstandard extraction method is to soak or immerse a sample or flush the fluid pathway withextracting fluid tha
38、t LRWhas per pair of gloves, for 40 to 60 min at a temperature between 37 to 40C. been heated to 37 6 1.0C,keeping the extracting fluid in contact with the relevant surface(s) for no less than 1 h.Alternate extraction or rinsing methods maybe used, but must be demonstrated to be equivalent or better
39、 than the standard method.6.2.3 The extraction should be performed in a way to ensure that all surfaces of the gloves that would have patient contact, comein contact with the extraction medium. For example, the glove may be lowered into a flask containing 40 mLsufficient volume ofLRWWater for BET wi
40、thfor the full exterior of the glove to be in complete contact with the water and the cuff (2 cm) folded overthe opening at the neck of the flask.flask and held in place by a non-pyrogenic stopper by the cuff.6.2.4 After the sample has been extracted, remove the glove. If appropriate, the second glo
41、ve of the pair can then beextractedExtracts or samples may be pooled for testing. For example, a glove may be extracted, removed and a second glove maybe suspended in the same flask with the extraction fluid of the first glove or extraction liquid. Alternately, both gloves may beextracted in the 40
42、mL LRW at the same time by both being suspended simultaneously in the flask, held in place by anon-pyrogenic stopper by the cuff at the opening of the same flask.6.2.5 Powder and other particulate matter can interfere with endotoxin determination assays. Interference should be overcomewith sample di
43、lution. Neither extract filtration nor centrifugation for clarification or the removal of particulates are acceptabletreatment methods as endotoxin can be coincidentally removed from the test sample.6.2.6 Test the sample by one of the methods identified in Section 7, Test Methods. If not tested imme
44、diately, the sample shouldbe frozen or refrigerated to prevent microbial growth, which willmay increase endotoxin levels.3 The boldface numbers in parentheses refer to the list of references at the end of this standard.TABLE 1 Sample SizeBatch Size # of Samples (Glove Pairs), Pyrogen Test (5).7.1.6.
45、2 Pyrogen Test InterpretationIf none of the temperatures rise more than 0.5C of the test animals respective base linetemperature, the glove meets the requirement for non-pyrogenic.7.2 Product Validation:7.2.1 The validity of the test results requires demonstration that the test article does not inhi
46、bit or enhance the LAL reaction.This is demonstrated through suitability testing for each product or product family by performing inhibition/enhancement testing.D7102 1747.2.2 The continued suitability of the assay should be re-assessed when significant changes are made that could adversely affectth
47、e accuracy of the BET result.8. Test Parameters8.1 Time:8.1.1 The gel-clot methods are typically incubated for 60 6 2 min.8.1.2 The kinetic methods measure the time it takes for a series of standards and unknown to read a pre-determined opticaldensity or color intensity.8.2 Temperature: Temperature8
48、.2.1 All BET methodologies are typically carried out at 37 6 1C.All BET methodologies are typically carried out at theincubation temperature recommended by the lysate manufacturer (typically 37 6 1C).8.3 pH: pHThe LAL test (a mixture of the sample with lysate) performs optimally at a pH between 6 an
49、d 8 pH units. Referto the lysate manufacturers package insert for further information and methods of adjustment.8.4 Product Validation:8.4.1 The validity of the test results requires demonstration that the test article does not inhibit or enhance the LAL reaction.8.4.1 For USP testing, three production lots of each product type should be tested for inhibition and enhancement separatelyfor a complete validation.Recovery:8.4.1.1 Kinetic Methods and Endpoint ChromogenicTurbidimetric and Chromogenic MethodsThe positive product control(
