1、Designation: E3002 15Standard Practice forAssessing the Comparative Efficacy of Products Used forthe Decontamination of Chemical Warfare Agents (CWAs)on Skin1This standard is issued under the fixed designation E3002; the number immediately following the designation indicates the year oforiginal adop
2、tion or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice establishes an in-vivo method for assessingthe comparative
3、 efficacy of products used for the decontami-nation of chemical warfare agents (CWAs) on the skin.1.2 This practice provides a quantitative efficacy compari-son of different skin decontamination products.1.3 To minimize the number of animals used, this in-vivopractice should be performed only after
4、rigorous in-vitrostudies of the candidate decontaminant, which can show theimplied claims including chemical neutralization, decontami-nation studies on surfaces and appropriate testing such ascytotoxicity.1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement
5、 are included in thisstandard.1.5 This standard does not purport to address all of thesafety concerns, if any, associated with the use of decontami-nation products or CWAs. It is the responsibility of the user ofthis standard to establish appropriate safety and health prac-tices and determine the ap
6、plicability of regulatory limitationsprior to use.2. Terminology2.1 Definitions of Terms Specific to This Standard:2.1.1 Chemical Warfare Agents (CWA), ntoxic chemicalsthat have been used as chemical weapons, or have beendeveloped for use as chemical weapons.2.1.1.1 DiscussionThe most common chemica
7、l warfareagents are: (1 and 2):2(a) nerve agentstabun (GA), sarin(GB), soman (GD), cyclosarin (GF), VX; and (b) blister agents(or vesicants)mustard and lewisite.2.1.2 decontamination, nthe process of physical removalor chemical neutralization, or both, of CWAs to decrease orprevent health effects du
8、e to a dermal contamination.2.1.3 in-vitro study, nstudy or protocol performed outsideof a living organism, either with or without the use of abiological material.2.1.4 in-vivo study, nstudy using a whole living organism.2.1.5 Organophosphate Agent (OP), nthe general namefor esters of phosphoric aci
9、d that are toxic through inhibition ofthe enzyme acetylcholinesterase.2.1.6 Protective Ratio (PR), nthe LD50of the decontami-nated animals divided by the LD50of the positive control(exposed to CWAs and not decontaminated) animals (3-5).2.1.7 vesicant agenta chemical agent that causes burnsand destru
10、ction of tissue.2.2 Acronyms:2.2.1 GAcommon name: Tabun; IUPAC name: (Ethyldimethylphosphoramidocyanidate): Organophosphate nerveagent.2.2.1.1 DiscussionThis nerve agent is the easiest to manu-facture. Consequently, it is more likely that developing coun-tries start their CW arsenal with this nerve
11、agent whereasindustrialized countries considerTabun to be out-of-date and oflimited use.2.2.2 GBcommon name: Sarin; IUPAC name: (RS)-Propan-2-yl methylphosphonofluoride) Organophosphatenerve agent.2.2.2.1 DiscussionGB is a volatile substance mainly takenup through inhalation.2.2.3 GDcommon name: Som
12、an; IUPAC name: (O-Pinacolyl methylphosphonofluoridate Organophosphate nerveagent.2.2.3.1 DiscussionA moderately volatile substance whichcan be taken up by inhalation or skin contact.2.2.4 GFcommon name: Cyclohexyl sarin; IUPAC name:(Cyclohexyl methylphosphonofluoridate) Organophosphatenerve agent.2
13、.2.4.1 DiscussionA substance with low volatility which1This practice is under the jurisdiction of ASTM Committee E54 on HomelandSecurity Applications and is the direct responsibility of Subcommittee E54.03 onDecontamination.Current edition approved June 15, 2015. Published June 2015. DOI: 10.1520/E3
14、002-15.2The boldface numbers in parentheses refer to a list of references at the end ofthis standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1is taken up through skin contact and inhalation of the substanceeither as a gas or ae
15、rosol.2.2.5 HDcommon name: Mustard or Distilled SulfurMustard; IUPAC name: (bis(2-chloroethyl) sulfide); Vesicant.2.2.5.1 DiscussionIn its pure state, mustard agent is col-orless and almost odorless.2.2.6 Lcommon name: Lewisite: IUPAC name: (2-chloroethenylarsonous dichloride). Vesicant.2.2.7 LD50a
16、standard measure of toxicity. The individualdose required to kill 50 % of the animals in a test population.2.2.8 VXcommon name: VX, IUPAC name: (O-ethylS-2-(diisopropylamino)ethyl methylphosphonothioate).2.2.8.1 DiscussionOrganophosphate nerve agent, a persis-tent substance which can remain on mater
17、ial, equipment andterrain for long periods. Update is mainly through the skin butalso through inhalation of the substance as a gas or aerosol.3. Summary of Practice3.1 Due to the extreme hazards of the chemical warfareagents, the efficacy of decontamination products cannot beevaluated in a human cli
18、nical study. This practice has beenused to support FDA clearance (for example, RSDL3(6 and7) 510k K023969) for decontamination devices for use onhuman skin (3-5).3.2 Determination of the efficacy of decontamination prod-ucts for use on the skin against these toxic compounds requiresin-vivo data, whi
19、ch are more physiologically relevant thanin-vitro studies. This practice provides a methodology forobtaining comparative in-vivo data.3.3 The practice is used in order to calculate the ProtectiveRatio of the decontaminant. The Protective Ratio is the LD50ofanimals treated with the chemical agent and
20、 decontaminated,divided by the LD50of control animals (animals treated withthe contaminant and not decontaminated) measured 24 hoursafter exposure to the CWA.3.4 This practice is based on decontamination efficacy ofdecontamination products against two nerve agents and oneblister agent, for a total o
21、f three CWAs. The nerve agents areeither G-agents or V-agents based on their chemical structures.The two nerve agents included in the practice are GD (fromG-agents) and VX (from V-agents). The blister agent includedin this practice is HD.4. Significance and Use4.1 This practice specifies an in-vivo
22、measurement of CWAdecontamination on the skin.4.2 CWA skin decontaminants will have different modes ofaction including absorption, adsorption, removal, chemicalneutralization or some combination of the above. There is,therefore, no single representative in-vitro method for valida-tion of decontamina
23、tion efficacy of products for skin decon-tamination. For example, measuring the presence of a radiola-belled chemical warfare agent after chemical neutralization,may give a false positive results. It has been shown that if theagent has been chemically neutralized, the radiolabel may stillbe present
24、in a non-toxic molecule. In addition, some chemicalneutralization methods may break down the original agent, butthe breakdown product is highly toxic. In the case of VX,hydrolysis produces a highly toxic product, EA2192 (S-(2-diisopropylaminoethyl) methylphosphonothioic acid (8).4.3 This standard pr
25、actice is of significance in that efficacyis thoroughly evaluated to the extent possible to represent useon human skin. In-vivo studies have demonstrated that simplechemical monitoring for disappearance of the chemical agentmay not be sufficient to measure decontamination and neutral-ization effecti
26、veness. A standard practice is needed for deter-mining actual decontamination and neutralization by measur-ing the decrease in mortality or lesion size caused by the agent.5. Reagents5.1 All the CWAs for these experiments shall be synthesizedin the laboratory where the experiments will be performed
27、orobtained from a legitimate external source which shall beincluded in the report.5.2 All test materials shall be at the same temperature as thatof the room where the test is conducted at the time ofapplication to the animals.5.3 Appropriate solvents shall be purchased from legitimatevendors as requ
28、ired and disclosed in the report.5.4 The standard decontaminant used for comparison shouldbe the product currently accepted for use by the majority ofDefense Forces and First Responders in the world, which atthis point in time is the RSDL skin decontamination system.6. Procedure6.1 A reputable labor
29、atory animal supplier must be used forany specified species or strain. Only a single constant source ofsupply for each species should be used by the testing laboratoryto maintain genetically homogenous test subjects.6.2 Animal CareAll animal care should conform to theappropriate standards (Associati
30、on for Assessment and Ac-creditation of LaboratoryAnimal Care InternationalAAALAC;http:/www.aaalac.org/) and all animal test protocols must haveappropriate approvals.6.3 As required by the Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC), animalcare and recording of the
31、 information including, but not limitedto, the following will be expected:6.3.1 Condition of the AnimalsOnly animals that show nosigns of ill health shall be used for these studies.6.3.2 Pre-test ConditioningAll animals to be used in thesestudies shall be quarantined for a designated period of timed
32、ependent on species and strain and source of the animal toensure good health prior to use. The period of quarantineshould be appropriate for the species and strain of animal. Anadequate diet for the species and fresh water shall be allowed3The sole source of supply of RSDL known to the committee at
33、this time isEmergent BioSolutions, 400 Professional Drive, Suite 400, Gaithersburg, MD,20879. If you are aware of alternative suppliers, please provide this information toASTM International Headquarters. Your comments will receive careful consider-ation at a meeting of the responsible technical comm
34、ittee,1which you may attend.E3002 152with free access. Each animal shall be identified, and foodconsumption should also be noted. Laboratory animal housingand test environmental conditions shall be maintained accord-ing to the acceptable animal care accreditation.Any significantdeviations shall be n
35、oted and reported.6.3.3 Weight of Test AnimalPretest weights of the testanimal shall be taken five days before and just prior toapplication of the test material. Only animals that eithermaintain their weight or show an increase equivalent to othersof the same age, sex, and starting weight shall be u
36、sed for thestudies. In the selection of the test subjects an attempt shouldbe made to produce as nearly identical groups as possible.6.3.4 Number of Test AnimalsAll of the studies designedto measure efficacy of decontaminants need two (2) sets ofanimals. The first set of animals is used to identify
37、thepercutaneous 24 hours LD50for nerve agents or the appropriatedosage range to create measurable lesion sizes. The second setof animals is used to test the comparative efficacy of thedecontamination products. For determining LD50values ofnerve agents without decontamination, 3 animals per dose, 5do
38、ses per agent will be tested. Thus for the two nerve agents(GD and VX), 3 animals per dose 5 doses per agent 2 nerveagents = 30 animals are required. For determining LD50valueswith decontamination, three stages, 1-2 animals per dose, 4-5doses will be tested using sequential stage wise methods (9 and
39、10). For the blister agent, 2 sets of animals 3 animals per set 1 blister agent = 6 animals required. All test groups shouldbe evenly divided by sex.6.3.5 Animal PreparationEach animal should be exam-ined 24 hours prior to testing. No animals showing signs of illhealth shall be sued for the testing.
40、 The experimenter shouldcarefully remove the hair from the backs and sides of the testanimals by closely clipping with an electric animal clipper, andtake care not to clip closely so as to abrade the skin. Based onpreliminary studies or past experience, establish an area on theskin of sufficient siz
41、e to allow application of the test material.6.3.6 Animal CareAnimals will be received into a vi-varium and supplied cage changes, food and water by animalcare staff until their day of treatment. Health checks and anymonitoring following treatment will be conducted by experi-mental staff. Animals wil
42、l be housed individually. Approvedstandard operating practices (SOPs) will be followed aboutReceiving Animals into Vivarium, Identification of Animals,Animal Husbandry Small Animals, Changing and Cleaningof Small Animal Cages, Lab Animal Feeds, and Shaving ofRodents. After 24 hours all test animals
43、will be euthanizedusing an appropriate method as included in Section 7.6.4 The animals may undergo an inhalation induction withisoflurane in a carrier gas at appropriate flow rate.6.5 A patient monitor shall be used to continually monitoranimals for physiological parameters such as heart rate, blood
44、pressure, arterial oxygen saturation, respiration rate, and tem-perature and these parameters shall be recorded.7. Test Methods7.1 This practice utilizes the clipped hair guinea pig model(3-5). With proper documentation, other animal models withskin similar to human skin, such as hairless guinea pig
45、, rabbit,minipig, or swine, can be used.7.2 Animals should be anaesthetized during the study.Anesthesia should be appropriate to the animal model selected.7.3 Exposure to the nerve agents is done by applying asingle dose to the application site 5 min after anesthesia. Ifneeded to improve dosing accu
46、racy, diluted agent may be used.NOTE 1Use caution when handling nerve agents.7.4 Exposure to the blister agent is done by applying threedoses to three application sites 5 min after anesthesia. Ifrequired, to improve dosing accuracy, diluted agent may beused.NOTE 2Use caution when handling blister ag
47、ents.7.5 Two minutes after agent application, the dosing siteshall be untreated (control) or decontaminated (per manufac-turer instructions for use) with the test and standard orcomparator decontamination product.7.6 Application method for the decontamination product isto be carefully thought out pr
48、ior to application to ensure thatmanufacturers instructions can be scaled down to the appro-priate treatment area.7.7 Determination of LD50of nerve agents a process whichis essentially identification of the dose response curve, shouldbe measured by using doses varying from non-lethal to lethalby est
49、ablishing 24 hour dose response curves in sets of animalsreceiving various doses of agent spanning the non-lethal tolethal dose range using sequential stage wise methods (9 and10). Randomly assign the animals to each treatment group andassess mortality at 24 hours. The LD50estimate value iscomplete when the difference between the upper and lower95 % confidence limits, divided by the LD50is 0.8 or less.7.8 To measure decontamination and neutralization efficacy,the study is repeated at agent concentrations starting slightlybelow the LD50a
