1、Designation: E 939 94 (Reapproved 2006)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 E 939; the number immediately f
2、ollowing 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 (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test me
3、thod 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 t
4、o determine the repellency of candidate compounds forother flying insects that attack humans.1.3 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 prac
5、tices and determine the applica-bility of regulatory limitations prior to use.2. Terminology2.1 Description of Term Specific to This Standard:2.1.1 complete protection time (CPT)the time from appli-cation of the repellent to the time of the first confirmed bite (asecond bite by the same species with
6、in 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 forearm or sometimes the lower leg. These areas are thenprotected from rubbing and are continuously exposed tomosquitoes in th
7、e 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 part of the final phaseof study in the development of mosquito repellents for personaluse.4.2 This test method is primaril
8、y 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 flexibility under arelatively wide range of circumstances and geographical loca-tions. By following this test method, internatio
9、nal 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 applications of arepellent for the following reasons:4.4.1 To determine how long the repellent is effective;4.4.2 To establish the e
10、ffective 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 terms of odor, stainingcapability, plasticizing effect, and oiliness or greasiness.4.5 No repellent should be tested on hum
11、ans without thewritten consent of the test volunteers (hereafter referred to astest subjects) and prior approval of competent authority, asdesignated in applicable laws and regulations governing ex-perimentation on humans.5. Apparatus5.1 Insect collection vials.5.2 Aspirator.5.3 Stereoscope (optiona
12、l).5.4 Standard References for Mosquito Identification, fordetermining species present in the field (optional).5.5 Temperature and Humidity Reading EquipmentIdeally, a continuous recording device such as a hygrothermo-graph should be used to record conditions during tests. If suchequipment is not av
13、ailable, readings should be made immedi-ately before, midway during, and immediately after the testsare made, using a sling psychrometer.5.6 Air Speed Indicator and Light Meter, optional butpreferred if equipment is available.5.7 Watch.5.8 Headnets.5.9 Cotton Gloves.1This test method is under the ju
14、risdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.12 on Insect Control Agents.Current edition approved April 1, 2006. Published April 2006. Originallyapproved in 1983. Last previous edition approved in 2005 as E 939 94 (2
15、005).1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.10 Battery-Operated Head Lamps, with red filters for testswith nocturnally active species.5.11 Notebook, Test Sheets, and Pencils, for recordingspecies, test data, date, and loca
16、lity 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 whether the clothingworn
17、 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 toxicological data may be re
18、quired.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 studies, formulations of l
19、otion,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 population (See Annex A1).7.2
20、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 Determine the identity of sp
21、ecies 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 studies with a 25 %etha
22、nol 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 if the CPTs are too s
23、hort 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 comparedirectly with anot
24、her 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 tested is not asimporta
25、nt 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 repellents and thestandar
26、d 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-cated by the CPT.8.8 Em
27、ploy 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 ofcandidate compound
28、s, 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 Because of their variab
29、ility 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, then Subject 1 will t
30、est 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 sufficient for a stati
31、sticalanalysis. 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 or more replications
32、of testswith each chemical on at least three different subjects arenecessary.8.11 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. Touching, rubbing, or abrasiveaction on
33、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 be the only known reason forloss in t
34、hese 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 will be reduced.E 939 94 (2006)28.14 I
35、ntermittent 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 effective means of testing
36、the 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 biting pressure tobe conc
37、entrated 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 pressure by exposing anun
38、treated 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 when test repellents ha
39、ve 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 head lamp, data sheet,
40、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 respective location an
41、d, 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-treatedforearms. Vials containing
42、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 exposureperiod, percent protecti
43、on provided by a repellent as comparedwith no treatment, and duration of protection against particularspecies.bites on untreated leg 2 bites on treated arm!4 bites on untreated leg 3 1009. Calculation9.1 For the balanced incomplete-block experiments, calcu-late an analysis of variance using methods
44、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 conditions.NOTE 8The foll
45、owing equation may be used to compute the adjustedaverage protection.3Average Ti52 Ti2 Birn1 Mwhere: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 = the number of
46、 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 ineffective.9.4 In
47、 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 the standard as
48、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-la
49、boratory test program and encourages interested parties tocontact the Committee E35 staff manager at ASTMHeadquarters.2Kempthorn, O., The Design and Analysis of Experiments, John Wiley and Sons,Inc., New York, NY, 1952, Section 26.4, p. 532.3Gilbert, I. H., Gouck, H. K., and Smith, C. N., “New Mosquito Repellents,”Journal of Economic Entomology, Vol 48, No. 6, December 1955, pp. 741743.E 939 94 (2006)3ANNEX(Mandatory Information)A1. SAMPLE WORK SHEETA1.1 The sample work sheet contains simulated field testdata to show how information is recorded and the CPT iscalculated.
