1、Designation: E981 04 (Reapproved 2012)Standard Test Method forEstimating Sensory Irritancy of Airborne Chemicals1This standard is issued under the fixed designation E981; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of
2、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 laboratory test method provides a rapid means ofdetermining sensory irritant potential of airborne chemicals ormi
3、xtures. It may also be used to estimate threshold limit values(TLV) for man. However, it cannot be used to evaluate therelative obnoxiousness of odors.1.2 This test method is intended as a supplement to, not areplacement for, chronic inhalation studies used to establishallowable human tolerance leve
4、ls.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 practices and determine the applica-bility of regulatory limitations prior to use. Specific ha
5、zardinformation is given in Section 6.2. Summary of Test Method2.1 This test method quantitatively measures irritancy asindicated by the reflex inhibition of respiration in mice exposedto sensory irritants.2.2 Four mice are simultaneously exposed to the airbornechemical. Usually a sufficient number
6、of groups of animals areexposed to a geometric series of concentrations so that aconcentration-response curve can be constructed. For simplepreliminary comparisons, however, a single group of fouranimals at one concentration will suffice.2.3 The mice are placed in a body plethysmograph attachedto an
7、 exposure chamber so that only the head is exposed to thetest material. The plethysmographs are connected to pressuretransducers, which sense changes created by inspiration andexpiration.The amplified signals are transmitted to a polygraphrecorder.2.4 The concentration of airborne irritant that prod
8、uces a50 % decrease in respiratory rate (RD50) is determined fromthe concentration-response curve constructed from the variousdata points obtained with a series of concentrations.3. Significance and Use3.1 This test method was developed to meet the followingcriteria:3.1.1 It provides positive recogn
9、ition of sensory irritants ofwidely varying potencies.3.1.2 It is sufficiently simple to permit the testing of largenumbers of materials.3.1.3 This test method is capable of generatingconcentration-response curves for purposes of compound com-parison.3.1.4 This test method has good reproducibility.3
10、.2 This test method can be used for a variety of divergentpurposes, including the assessment of comparative irritancy ofcompounds or formulations and setting interim exposure levelsfor the workplace (1, 2).23.3 It has been shown that for a wide variety of chemicalsand mixtures, a perfect rank order
11、correlation exists betweenthe decreases in respiratory rate in mice and subjective reportsof sensory irritation in man (1, 3, 4, 5).3.4 Aquantitative estimate of the sensory irritancy of a widevariety of materials can be obtained from concentration-response curves developed using this method (1, 3,
12、4, 6, 7, 8,9).3.5 Although this test method is intended to measure sen-sory irritation of the nasal mucosa, the cornea is innervated bythe same nerve. This animal model will, therefore, allow anestimate of the irritant potential of cosmetic ingredients orother household products to the eye, assuming
13、 that they can beaerosolized (10).3.6 This test method is recommended for setting interimguidelines for exposure of humans to chemicals in theworkplace, to assess acute sensory irritation resulting frominadvertent spills of household products, and to assess thecomparative irritancy of formulations o
14、r materials intended fora variety of uses (see Appendix X2).3.7 This test method will detect irritating effects at concen-trations far below those at which pathological changes areobserved (9).1This test method is under the jurisdiction of ASTM Committee E47 onBiological Effects and Environmental Fa
15、te and is the direct responsibility ofSubcommittee E47.02 on Terrestrial Assessment and Toxicology.Current edition approved Dec. 1, 2012. Published December 2012. Originallyapproved 1984. Last previous edition approved in 2004 as E981 04. DOI:10.1520/E0981-04R12.2The boldface numbers in parentheses
16、refer to the list of references at the end ofthis standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1NOTE 1A good overview of the toxicological evaluation of irritantcompounds is given in Ref (8).4. Apparatus4.1 The apparatus re
17、quired to perform this test is listedbelow. The basic components for testing any type of materialare the same.Alist of suitable apparatus and suppliers is foundin Appendix X1.4.2 Plethysmograph Tubes.4.3 Exposure Chamber, constructed entirely of glass, with avolume of 2.3 L.4.4 S.T.103/60 Ground Gla
18、ss Joint, that allows access to theinside of the exposure chamber.4.5 Perforated Rubber Dental Dam, reinforced with electri-cal tape.4.6 Rubber Stoppers.4.7 “T” Tube, with a tube 6 cm long and the “T” 12 cm long.4.8 Vacuum Pump.4.9 Flowmeter.4.10 Absolute Filter.4.11 Sodium Carbonate-Activated Charc
19、oal Filter.4.12 Pressure Transducer.4.13 Polygraph Recorders.4.14 Frequency-to-Voltage Converter, operating in the aver-aging mode instead of the pulse mode. See Appendix X1.7.4.15 Voltage Addition and Division Equipment, to obtain thesignal average for four mice.4.16 Signal Averages.4.17 Oscillogra
20、ph.4.18 Aerosol Generator.4.19 Timer.4.20 Control Valve.5. Reagents5.1 Technical reagents may be used in all tests wheresolvents other than water are required.5.2 Solutions containing 1 to 3 % of the test material areused for comparative studies.6. Hazards6.1 Not all compounds that cause a decrease
21、in respiratoryrate are sensory irritants. To be characterized as a sensoryirritant, a compound must produce a net decrease in respiratoryrate as a result of the characteristic pause during expiration asNOTE 1Taken from Ref. (3).FIG. 1 Typical Tracing of Normal Mouse Respiration (Top), and of a“ Mode
22、rate” Sensory Irritant Response (Bottom)NOTE 1Taken from Ref. (8).FIG. 2 Typical Tracing of Normal Mouse Respiration (Top), a Mod-erate Pulmonary Irritant Response (Center), and an Extreme Pul-monary Irritant Response (Bottom)E981 04 (2012)2shown in Fig. 1. This pause differentiates sensory irritant
23、s frompulmonary irritants, general anesthetics, and asphyxiants,which also reduce respiratory rate, but as a result of a pausebetween breaths as shown in Fig. 2.6.2 It is possible for one component to alter the effect ofanother in a mixture, depending on their respective concentra-tions (11). Additi
24、ve and antagonistic responses are possible.For this reason the effects of each compound in a formulationshould be assessed before any test is made for interactions.6.3 Although the test procedure has been found to show ahigh correlation for sensory irritants with established TLVvalues for man, it ma
25、y well predict values that are too high forcompounds of low reactivity that are metabolically activated,and also for pulmonary irritants (10).7. Test Animals7.1 Mice are the subjects to be used for this test. It isimperative that they meet the specifications outlined here.Although any mouse of the p
26、roper size could be used, markeddifferences have been observed between different strains andsexes (2).7.1.1 Male Swiss-Webster mice shall be used as the testsubjects.7.1.2 Only animals weighing between 22 and 28 g may beused. Smaller mice might be able to crawl into the exposurechamber, while larger
27、 ones may not be able to breathe normallyin the apparatus.7.1.3 The same system can be used with guinea pigs or ratswith an airflow of 2 L/min when using head dome (9).8. Preparation of Apparatus8.1 Exposure Chamber:8.1.1 The heads of each of four mice extend into theexposure chamber, and the bodies
28、 are contained in plethysmo-graph tubes. Perforated rubber dental dam reinforced with electrical tape provides tight but comfortable seals around theNOTE 1Dimensions are in centimetres.NOTE 2Taken from Ref. (19).FIG. 3 Glass Exposure Chamber with Attached Body PlethysmographsNOTE 1Taken from Ref. (1
29、9).FIG. 4 Diagram of Test ApparatusE981 04 (2012)3animals necks, and rubber stoppers prevent them from back-ing out of the tubes, and provides an airtight body plethysmo-graph (see Fig. 3).8.1.1.1 The “T” tube is of the same diameter as the inlet tothe chamber. The gas or aerosol from the generator
30、enters oneside of the “T” and the makeup air enters on the other. Thus thetube acts as a miniature mixing chamber, eliminating the needfor a baffle plate. The “T” tube is not shown in Fig. 3.8.1.2 Chamber Equilibration:8.1.2.1 It is desirable to reach equilibrium of the testmaterial in the exposure
31、chamber in as short a time as possible.In no case should this time exceed one-tenth of the totalexposure time. The validity of the data for extrapolation to manrequires rapid attainment of maximum concentration.8.1.2.2 Equilibration time in minutes is 5.0 times the cham-ber volume in litres divided
32、by airflow through the chamber inlitres per minute (12).8.2 A vacuum pump with a control valve monitored by aflowmeter provides a constant airflow through the exposurechamber. Chamber effluent is passed through an absolute filterand then a sodium carbonate-activated charcoal filter beforeexhausting,
33、 preferably into a fume hood. (See Fig. 4.)8.3 Each of the four plethysmograph tubes is connected to apressure transducer. As the mouse inhales, a positive pressureis created and exhalation results in a negative pressure. Theamplified signals are recorded on a polygraph, which has thepolarity set so
34、 that an upward deflection is obtained duringinspiration and a downward deflection is obtained duringexpiration. The signal from each transducer is also fed into afrequency-to-voltage converter, and then fed into a signalaverager. The output of the averager is displayed on a secondrecorder, thus per
35、mitting continuous monitoring of the averagerespiratory rate of the four mice. (See Fig. 4.)8.4 A suitable generator for this test is a glass Dautrebande-type generator modified to allow continuous feed of testmaterial.3This generator can be used for volatile or nonvolatileliquids, solutions, or sus
36、pensions of solids. It is depictedschematically in Fig. 5.8.4.1 For aqueous solutions, liquid is delivered via a pumpregulated at 1.0 mL/min to the right-hand tube. This deliveryrate can be varied by a factor of 3 to 4. Air is delivered at 10to 12 psig when a water solution is used, and 8 to 10 psig
37、 whenacetone solution is used. With acetone the amount of solutiondelivered is restricted so that no more than 3000 ppm acetonevapor is produced in the exposure chamber. The calculation ismade from the total airflow used in the chamber. At thestandard flow rate of 20 L/min through the chamber, deliv
38、eryto the generator of 0.22 mL of acetone per minute will result ina concentration of 2800 to 3000 ppm. With acetone there willbe no liquid overflow, but with aqueous solutions, 1.0 mL/minis high enough so that liquid will fall to the bottom of thegenerator. This is collected in a reservoir via the
39、overflow tube.8.4.2 Arrows in Fig. 5 indicate the path that the aerosol willfollow. Polyethylene Glycol 200 (PEG 200) can be used as asolvent instead of water. The air pressure should be about 20 to25 psig with this solvent. Dry air must be used with PEG 200,which is hygroscopic. Using this generato
40、r with a 1 % solutionof test material in water and 20 L/min flow rate through theexposure chamber, the concentration in the chamber will bebetween 10 to 20 mg/m3and most particles will be submi-cronic.8.4.3 The Dautrebande-type generator can also be used tovaporize liquids for exposure of animals to
41、 vapors. For thispurpose, the liquid is delivered at a known rate by a regulatedpump and airflow is set at 10 to 20 psig. For liquids of lowervapor pressure, heating tape can be used around the generatorto increase vaporization efficiency. For aerosols or vaporslikely to oxidize rapidly in air, dry
42、nitrogen should be usedinstead of air. When this is done, pure oxygen is added to thechamber airflow to maintain 18 to 20 % O2in the exposurechamber. When suspensions are to be tested, the suspendedmaterial must be very fine to prevent clogging of the tip on thegenerator. Although larger tips can be
43、 used if required, adegradation of aerosolizing performance will result from theiruse.3Pitt No. 1 aerosol generator available from Scientific Glassblowing Laboratory,McKees Rocks, PA 15136, has been found suitable.NOTE 1Taken from Ref. (12).FIG. 5 Schematic Representation of the Pitt No. 1 Aerosol G
44、en-eratorE981 04 (2012)48.5 To start and stop test material generation, a timer and anassociated control valve are needed in conjunction with theaerosol generator.8.6 When using water or acetone a “dry” particle will beproduced, since both solvents will evaporate. However, PEG200 will not evaporate
45、and a liquid droplet is obtained. Massconcentration in the chamber should be obtained by samplingon filters and weighing on an appropriate balance. A bettermethod, but one not required in a screening experiment, isappropriate chemical analysis. When acetone is used, itsconcentration in the chamber s
46、hould be verified. Indicator tubeanalysis is adequate, or an infrared analyzer or gas chromato-graphic analysis can be used.8.7 Gases are delivered directly into the exposure chambervia an appropriate flowmeter.8.8 With the exception of the exposure chamber which isessentially a unique piece of appa
47、ratus, other parts can besubstituted by similar equipment. Also, minicomputers can beused to replace the frequency-to-voltage converter and signal-averaging device. The magnetic tape is not required, and afour-trace oscilloscope with storage capability can replaceoscillograph No. 1.9. Sample Prepara
48、tion9.1 Because of the large variety of chemicals and formula-tions that can be tested by this procedure, and the tremendousdifferences in irritant potential between them, no specificstipulation for sample preparation can be made. The onlyrequirement for concentration is that the levels to be tested
49、 arespaced at even logarithmic intervals to allow goodconcentration-response curves to be generated from the dataobtained. The information provided in the succeeding para-graphs of this section is therefore intended for general guidanceonly.9.2 For solids and nonvolatile liquids, solutions are pre-pared in an appropriate solvent. Water and polyethylene glycol200 (PEG 200) are the most commonly used for this purpose,although 0.1 N HCl, 0.1 N NaOH, and acetone can also beused. In the case of acetone, which is a mild irritant, theconcentration in the chamber should be kept be
