1、Designation: E939 94 (Reapproved 2012)Standard Test Method ofField Testing Topical Applications of Compounds asRepellents for Medically Important and Pest Arthropods(Including Insects, Ticks, and Mites):I Mosquitoes1This standard is issued under the fixed designation E939; the number immediately fol
2、lowing 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 () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test metho
3、d is used to evaluate the repellency ofpromising compounds that have undergone primary laboratorystudies and have been approved for skin application forsecondary testing.1.2 This test method is designed for the study of mosquitorepellents, but with some modifications this test method can beused to d
4、etermine the repellency of candidate compounds forother flying insects that attack humans.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns, if any, assoc
5、iated 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. Terminology2.1 Definitions of Terms Specific to This Standard:2.1.1 complete protection time (CPT)
6、the time from appli-cation of the repellent to the time of the first confirmed bite (asecond bite by the same species within 30 min of the first). Thispermits any number of unconfirmed bites during the CPT.3. Summary of Test Method3.1 A measured amount of the candidate material is appliedto the fore
7、arm or sometimes the lower leg. These areas are thenprotected from rubbing and are continuously exposed tomosquitoes in the field to determine the length of time thetreatment provides either complete protection or a high level ofprotection.4. Significance and Use4.1 This test method is an important
8、part of the final phaseof study in the development of mosquito repellents for personaluse.4.2 This test method is primarily designed to simulate asituation in which a person treated with a repellent is exposedto natural populations of attacking mosquitoes.4.3 The simplicity of the test offers flexib
9、ility under arelatively wide range of circumstances and geographical loca-tions. By following this test method, international testing witha variety of vector mosquito populations is no more difficult toaccomplish than tests with various domestic species.4.4 A number of people test topical applicatio
10、ns of arepellent for the following reasons:4.4.1 To determine how long the repellent is effective;4.4.2 To establish the effective dosage range;4.4.3 To establish the range of effectiveness on severalmosquito genera and species in a number of geographicalareas; and4.4.4 To identify the material in t
11、erms of odor, stainingcapability, plasticizing effect, and oiliness or greasiness.4.5 No repellent should be tested on humans without thewritten consent of the test volunteers (hereafter referred to astest subjects) and prior approval of competent authority, asdesignated in applicable laws and regul
12、ations governing ex-perimentation on humans.5. Apparatus5.1 Insect collection vials.5.2 Aspirator.5.3 Stereoscope (optional).5.4 Standard References for Mosquito Identification, fordetermining species present in the field (optional).5.5 Temperature and Humidity Reading EquipmentIdeally, a continuous
13、 recording device such as a hygrothermo-graph should be used to record conditions during tests. If such1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommittee E35.12 on Insect Contr
14、ol Agents.Current edition approved April 1, 2012. Published June 2012. Originallyapproved in 1983. Last previous edition approved in 2006 as E939 94 (2006).DOI: 10.1520/E0939-94R12.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesNOTICE
15、: This standard has either been superseded and replaced by a new version or withdrawn.Contact ASTM International (www.astm.org) for the latest information1equipment is not available, readings should be made immedi-ately before, midway during, and immediately after the testsare made, using a sling ps
16、ychrometer.5.6 Air Speed Indicator and Light Meter, optional butpreferred if equipment is available.5.7 Watch.5.8 Headnets.5.9 Cotton Gloves.5.10 Battery-Operated Head Lamps, with red filters for testswith nocturnally active species.5.11 Notebook, Test Sheets, and Pencils, for recordingspecies, test
17、 data, date, and locality of the test. A sample worksheet is attached with recorded results (see Annex A1.).5.12 Water Supply, Mild Soap, and Paper Towels, forwashing treated skin.5.13 Clothing, should be appropriate to the season andgeographical area.NOTE 1There are differences of opinion as to whe
18、ther the clothingworn should be uniform in color and type, however, data available do notreject or confirm this contention.5.14 List of Chemical Names, identifying the compounds ormixtures, or both, to be tested.NOTE 2In the event of a medical emergency, the chemical list alongwith pertinent toxicol
19、ogical data may be required.6. Reagents and Materials6.1 All test solutions are formulated on a weight to volumebasis (usually with 95 % ethanol).6.2 Test chemicals in 25 % ethanol solutions.6.3 Test standard deet (N,N-diethyl-meta-toluamide) in a25 % ethanol solution.6.4 For final development studi
20、es, formulations of lotion,liquids, creams, solids, or pressurized aerosols containingingredients for field evaluation are included.7. Sampling7.1 Each candidate repellent is paired with each otherrepellent or a standard on the arms of a subject and exposedsimultaneously to the same insect populatio
21、n (See Annex A1).7.2 A round-robin or paired test is used in the experiments,usually based on the number of experimental materials beingevaluated.7.3 Treatments are exposed to the mosquito population foras long as the repellents are effective, and the biting activitycontinues.8. Procedure8.1 Determi
22、ne the identity of species of mosquito in the testarea prior to the test. Determine the time to begin and end testseach day by the activity of the species to be tested. Diurnalnocturnal or other patterns of mosquito behavior will governthe scheduling of tests.8.2 For field tests, make the initial st
23、udies with a 25 %ethanol solution of the candidate repellents (250 mg AI/mL). Acompound or formulation may be retested at reduced orincreased concentrations, or at full strength, if warranted.NOTE 3If the complete protection times (CPTs) of repellents are toolong for the available testing period or
24、if the CPTs are too short forevaluation, adjust the concentrations accordingly. The termination of anevaluation before a confirmed bite occurs is termed a “plus out” andshould be avoided.8.3 Spread 1 mL of the repellent formulation or repellentsolution evenly over the forearm of the subject and comp
25、aredirectly with another repellent of the same concentration on theother arm (see Note 4). The behavior of some species neces-sitates the use of the legs instead of the arms as treatment sites.This can be determined by observation before the tests begin.NOTE 4The concentration of a compound being te
26、sted is not asimportant as the assurance that it is paired with another compound ofequal concentration.8.4 If legs are used as treatment sites, apply 1.5 mL on theskin between the ankle and knee.8.5 Determine the surface area of the limbs of each testsubject so that treatment rates of candidate repe
27、llents and thestandard are uniformly applied. Adjust the application rates fordifferences in arm or leg size of different subjects.8.6 Expose the treated arms or legs continuously to naturalpopulations of the mosquito species being tested.8.7 The duration of effectiveness of the repellent is indi-ca
28、ted by the CPT.8.8 Employ a balanced incomplete block (BIB) experimen-tal design (round robin) when three to five chemicals are to betested. With this design, test each repellent in the series onopposite arms of a given number of subjects.NOTE 5Ideally, the number of subjects should equal the number
29、 ofcandidate compounds, excluding the standard. Thus, if four compoundsare to be tested, including the standard, three test subjects would berequired. To illustrate, the pairings would be: AB, AC, AD, BC, BD, andCD. Subject 1 would test AB and CD; Subject 2, AC and BD, and Subject3, AD and BC.8.9 Be
30、cause of their variability in attractiveness, assignrepellents to test subjects in a randomized and balanced fashionso that each subject does not wear the same combination ofmaterials more than once in a single BIB test series. Forexample, if compounds A, B, C, D, and E are tested with astandard F,
31、then Subject 1 will test AB, CD, and EF; Subject 2will test AC, BE, and DF; Subject 3 will test AD, BF, and CE;Subject 4 will test AE, BC, and CF; and Subject 5 will test AF,BC, and DE.NOTE 6If four or five compounds, excluding the standard, are testedin a BIB series, one replication of the BIB is s
32、ufficient for a statisticalanalysis. If three compounds are to be tested in this fashion, tworeplications of the round robin will be necessary.8.10 A direct comparison of the candidate versus the stan-dard repellent deet is used when fewer than three chemicals areto be tested. For these tests, four
33、or more replications of testswith each chemical on at least three different subjects arenecessary.E939 94 (2012)28.11 Express the results obtained by paired tests or BIBcomparisons at CPT in minutes or hours.8.12 During the experiment, subjects shall avoid contactwith the treated skin surfaces. Touc
34、hing, rubbing, or abrasiveaction on the treated skin can affect the results. Avoid unduesweating or wetting of the treated skin except in special testsdesigned to study the durability of repellents under adverseconditions. Loss due to evaporation and absorption is, ofcourse, unavoidable but should b
35、e the only known reason forloss in these tests, if the above precautions are taken.8.13 Test each species with its biting behavior in mind. Forexample, if a test subject remains immobile, some diurnallyactive mosquito species become less aggressive and bitingpressure on the repellent treated skin wi
36、ll be reduced.8.14 Intermittent walking, standing, and squatting, as wellas raising and lowering the arms periodically, are very effectivein attracting many of the daytime biting species. Test supervi-sors and subjects need to be aware of these behavioraldifferences so they will find the most effect
37、ive means of testingthe repellents against each of the various mosquito speciesstudied. To be successful, tests should be designed to accom-modate the test species rather than the investigator.8.15 Protect exposed parts of the body, such as the handsand face, by gloves and headnet in order for bitin
38、g pressure tobe concentrated on the treated skin and for the comfort of thesubjects.8.16 One responsible person records data and providesleadership to ensure the accuracy and uniformity of theexperiments.8.17 Use the number of bites per unit of time to indicatebiting pressure. Determine biting press
39、ure by exposing anuntreated arm (or leg) and counting the number of each speciesbiting in 1 min.8.18 Make biting counts before, periodically during, andafter each day of testing. Designated control subjects may beused to take biting rate counts. Participants in tests may makecounts between tests whe
40、n test repellents have been removedfrom the skin after failure to repel.8.19 Record whether the test chemical or repellent formu-lation has any of the objectionable characteristics mentioned in4.4.NOTE 7Identification of the species biting after dark is very difficult;therefore, in addition to a hea
41、d lamp, data sheet, and collection vials, eachtest subject has a folding seat and battery-powered aspirator. Seats arepositioned at approximately 10 m apart with the test subjects backs to theprevailing wind.8.20 To determine biting rate and species present, eachtreated subject sits at his or her re
42、spective location and, at onsetof mosquito activity at dusk and at 1 h intervals thereafter,aspirates mosquitoes biting the untreated lower legs from kneeto ankle for 1 min.8.21 Tests begin with the individuals instructed to use thevials to collect each mosquito biting their repellent-treatedforearm
43、s. Vials containing mosquitoes are placed sequentiallyin a compartmented flat so that later they can be matched witha record of the treatment and time of bite kept by each testsubject.8.22 The procedure makes possible the determination ofspecies biting, total numbers of bites during the exposureperi
44、od, percent protection provided by a repellent as comparedwith no treatment, and duration of protection against particularspecies.bites on untreated leg 2 bites on treated arm!bites on untreated leg 31009. Calculation9.1 For the balanced incomplete-block experiments, calcu-late an analysis of varian
45、ce using methods described in TheDesign and Analysis of Experiments.29.2 From this analysis of variance, determine the leastsignificant difference (5 % level) between any two repellents.9.3 Compute an adjusted average protection time that com-pensates for variation between hosts and testing conditio
46、ns.NOTE 8The following equation may be used to compute the adjustedaverage protection.3Average Ti52 Ti2 Birn1Mwhere:Ti= the total CPTs for all tests with repellent i,Bi= the total CPT for both repellents in all pairs in which therepellent i occurred,r = the number of times each pair was replicated,n
47、 = the number of repellents, andM = the grand mean of all CPTs in the series.Due to the nature of the statistical analysis, the adjusted mean mayoccasionally fall outside the CPT range and at times be a negative value.Such data occur only when the candidate repellent has a very low CPT andthus is in
48、effective.9.4 In the direct comparison tests between a candidaterepellent and a standard, use a paired t-test to determine if thereare significant differences (5 % level) between the standard andthe candidate.9.5 Express the relative duration of effectiveness of theexperimental repellent to that of
49、the standard as the ratio of theCPT.9.6 The ratio is a more reliable index than the actual CPTwhich may vary considerably between hosts and differentpopulations of mosquitoes.10. Precision and Bias10.1 No precision data is available for this test method,however, the committee is interested in conducting an inter-laboratory test program and encourages interested parties to2Kempthorn, O., The Design and Analysis of Experiments, John Wiley andSons, Inc., New York, NY, 1952, Section 26.4, p. 532.3Gilbert, I. H., Gouck, H. K., and Smith, C. N., “New Mosquito Repelle
