ASTM E981-19

Designation: E981 − 19Standard 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 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 ormixtures. 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 levels.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, health, and environmental practices and deter-mine the applicability of regulatory limitations prior to use.Specific hazard information is given in Section 6.1.4 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.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 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 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 produces 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 recognition 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.3.2 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 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, 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 that they can beaerosolized (10).3.6 This test method is recommended for setting interimguidelines for exposure of humans to chemicals in the1This test method is under the jurisdiction of ASTM Committee E50 onEnvironmental Assessment, Risk Management and Corrective Action and is thedirect responsibility of Subcommittee E50.47 on Biological Effects and Environ-mental Fate.Current edition approved Feb. 1, 2019. Published March 2019. Originallyapproved 1984. Last previous edition approved in 2012 as E981 – 04(2012). DOI:10.1520/E0981-19.2The boldface numbers in parentheses 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 StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1Copyright by ASTM Int l (all rights reserved); Mon Mar 18 02:11:43 EDT 2019Downloaded/printed byKongju National University (Kongju National University) pursuant to License Agreement. No further reproductions authorized.workplace, to assess acute sensory irritation resulting frominadvertent spills of household products, and to assess thecomparative irritancy of formulations or 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).NOTE 1—A good overview of the toxicological evaluation of irritantcompounds is given in Ref (8).4. Apparatus4.1 The apparatus required 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 Glass 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 Charcoal 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 Oscillograph.4.18 Aerosol Generator.4.19 Timer.4.20 Control Valve.NOTE 1—Taken from Ref. (3).FIG. 1 Typical Tracing of Normal Mouse Respiration (Top), and of a“ Moderate” Sensory Irritant Response (Bottom)NOTE 1—Taken 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 − 192Copyright by ASTM Int l (all rights reserved); Mon Mar 18 02:11:43 EDT 2019Downloaded/printed byKongju National University (Kongju National University) pursuant to License Agreement. No further reproductions authorized.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 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 asshown in Fig. 1. This pause differentiates sensory irritants 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 (12). Additive 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 may 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 proper size could be used, markeddifferences have been observed between different strains andsexes (2).NOTE 1—Dimensions are in centimetres.NOTE 2—Taken from Ref. (11).FIG. 3 Glass Exposure Chamber with Attached Body PlethysmographsNOTE 1—Taken from Ref. (11).FIG. 4 Diagram of Test ApparatusE981 − 193Copyright by ASTM Int l (all rights reserved); Mon Mar 18 02:11:43 EDT 2019Downloaded/printed byKongju National University (Kongju National University) pursuant to License Agreement. No further reproductions authorized.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 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 are contained in plethysmo-graph tubes. Perforated rubber dental dam reinforced withelectrical tape provides tight but comfortable seals around theanimals’ 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 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 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 by airflow through the chamber inlitres per minute (13).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, 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 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 permitting 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 suspensions 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 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, deliveryto 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 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 generator 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.3Pitt No. 1 aerosol generator available from Scientific Glassblowing Laboratory,McKees Rocks, PA 15136, has been found suitable.NOTE 1—Taken from Ref. (13).FIG. 5 Schematic Representation of the Pitt No. 1 Aerosol Gen-eratorE981 − 194Copyright by ASTM Int l (all rights reserved); Mon Mar 18 02:11:43 EDT 2019Downloaded/printed byKongju National University (Kongju National University) pursuant to License Agreement. No further reproductions authorized.8.4.3 The Dautrebande-type generator can also be used tovaporize liquids for exposure of animals to 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 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 used if required, adegradation of aerosolizing performance will result from theiruse.8.5 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 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