CROSS-ADAPTATION BY STRUCTURAL ANALOGS 367 0.6% in the E, SE form. GC/MS of the (Z)-ethyl-3-methyl-2-hexenoate showed 93.4% in the Z, SZ form, with the closely eluting exo compound, ethyl-3-methylidine-hexanoic ester (k'•x o = 3.5 ot = 1.05) comprising 5.4% of the sample. The remaining 1.2% exhibited a M + ion at m/z 128, indicating the presence of the free acid. This required additional chromatographic cleanup on the Zorbax Sil column to remove the acid and yield a pure ester sample 94% Z, SZ form 6% exo isomer. Nuclear magnetic resonance (NMR). A Varian XL 300 MHz instrument was employed for HNMR and CMR experiments with tetramethylsilane (TMS) as an internal reference. Resonances are given in delta units as measured downfield from TMS, and are given as: singlet (s), droplet (d), triplet (t), quartet (q), and multipier (m): (E) Ethyl-3-methyl-2-hexenoate: Mass spectrum: m/z (tel. int.) 41(56), 43(37), 53(17), 55(61), 67(17), 69(51), 82(43), 83(21), 84(3), 95(27), 100(37), 111(100), 113(53), 127(5), 128(38), 129(3), 141(10), M+156(43), 157(4). •HNMR: 5.17 (s, ]H), 4.15 (q,J = 7.2 Hz, 2H), 2.14 (s, 3H), 2.10 (dJ = 7.2 Hz, 2H), 1.51 (mJ = 7.2 Hz, 2H), 1.31 (t J = 7.2 Hz, 3H), 0.93 (t J = 7.2 Hz, 3H) •3C NMR: 162.0, 160.058, 115.551, 59.422, 42.931, 20.513, 18.615, 14.316, 13.629. (Z) Ethyl-3-methyl-2-hexenoate + ethyl-3-methylidene-hexanoic ester: •HNMR: 5.17 (s, 1H), 4.18 (q,J = 3.6 Hz, 2H), 4.17 (q,J = 7.2 Hz, 2H), 2.66-2.58 (m, 2H), 2.24-2.14 (m, 2H), 1.88 (s, 3H), 1.60-1.44 (m, 2H), 1.28 (t J = 7.2 Hz, 3H) •3CNMR: 160.533,116.128, 113.660, 59.407, 59.371, 35.215, 30.711, 25.412, 25.127, 21.418, 14.286, 14.075, 11.972 Mass spectrum: Z-ethyl-3-methyl-2-hexenoate: m/z (rel. int.)41(96), 43(81), 45(14), 53(37), 55(93), 67(33), 69(67), 81(21), 82(43), 83(17), 95(41), 99(4), 100(17), 111(100), 113(56), 127(4), 128(14), 141(13), M + 156(46), 157(6). Mass spectrum: Ethyl-3-methylidene-hexenoic ester: m/z (rel. int.)41(69), 43(64), 45(10), 51(9), 53(43), 55(100), 67(26), 69(33), 71(15), 81(36), 82(18), 83(13), 99(60), 100(4), 109(11), 110(22), 111(84), 113(6), 127(6), 128(22), 141(0.6), M+156(50), 157(5). STIMULI PRESENTATION The stimuli used are presented in Figure 1. The 3-methyl-2-hexenoic acids were syn- thesized and purified by column chromatography in a manner described previously (10). Both the acids and esters used as odorants were diluted in odorless, light, white, mineral oil and presented in 270-ml, polypropylene squeeze-bottles with plastic, flip-top caps. Each bottle contained 10 ml of the odorant/mineral oil solution. For the cross-adaptation procedure, the odorants used were a 10:1 (E)- to (Z)-3M2H mixture, a 10:1 (Z)- to (E)-3M2H mixture, and the individual isolated (E)- and (Z)- isomers of EE3M2H. A 12-step binary dilution series was prepared for each odorant. Initially, 20 mg of each isomeric mixture was diluted in 20 ml of odorless, light, white, mineral oil to yield a 1 mg/ml (0.1% w/v) solution with molar concentrations of 7.81 mM of 3M2H, 6.32 mM of EE3M2H, 5.37 mM of EE3M20, and 6.93 mM of EE3M2P. The dilution scheme for each odorant was the same, ranging from 1.0 x 10 •% w/v (step 12) to 4.88 x 10-5% w/v (step 1). For the threshold procedure, the odorants used were the individual (E)- and (Z)-isomers of EE3M2H and a 3:1 (E) to (Z) ratio of EE3M2H. A 24-step binary dilution series was prepared for each odorant, starting at 1.0 x 10 •% w/v (step 24). Cross-adaptation procedure. For each of the 3M2H mixtures, subjects were tested in two 30-minute sessions, which were separated by at least 24 hr. A forced-choice, staircase

368 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS procedure was used at the beginning of each session to equate intensities of the test stimuli. Each trial consisted of a step-10 concentration of the odorant to be used for adaptation and an alternating concentration of the other test odorant (starting at step 8). Subjects were instructed to identify the more intense of the two bottles. Each pair of stimuli was presented twice, with trials separated by one minute. If the subject selected the soon-to-be-adapting odorant as being stronger in each of the two trials, the subse- quent trial used the next stronger concentration of the other test odorant. Similarly, if the subject selected the other test odorant as stronger in each of the two trials, a weaker concentration of that odorant was used for the subsequent pairing. The concentration at which subjects failed to identify the same stimulus as stronger in two consecutive trials was selected as the concentration most similar in intensity to step 10 of the odorant to be used as the adapting stimulus. Thus, the test odorants were step 10 of the odorant for adaptation and the concentration of the other odorant judged to be most similar in intensity by the individual subject the stimulus used for adaptation was a fourfold higher concentration (i.e., step 12) than the test stimulus. A two-minute rest was imposed following perceptual matching. Subjects then rated, using magnitude estimation, the intensities of step 10 of the adapting stimulus and the intensity-matched concentration of the other test odorant. Subjects assigned numerical ratings to each of these two stimuli twice. If the means of the magnitude estimates for each odor were dissimilar (greater than 20% discrepancy), the matching procedure was repeated. In this manner, initial magnitude estimates ensured that the two stimuli were perceptually equivalent for that subject. After making the initial magnitude estimates, subjects began to sniff repeatedly the adapting stimulus. Every 15 seconds during this adaptation period, subjects sniffed and rated a test stimulus between sniffs of the adapting stimulus. The test stimulus, either 3M2H or one of the individual ethyl esters, alternated in sequential trials so that subjects made a total of 20 ratings (10 3M2H, 10 ethyl ester) during the 5-rain adaptation period. Following these ratings, the adapting stimulus was removed and subjects con- tinued to rate test stimuli every 15 seconds during a 5-rain postadaptation period. Subjects thus made a total of 20 ratings during this recovery period. In the second session, the adapting odorant, either 3M2H or one of the ethyl esters, was reversed and the entire procedure was repeated. The adapting odorant used in a par- ticular session was counterbalanced across sessions for all subjects. Each magnitude estimate was converted to a percentage of the initial magnitude esti- mate for that odorant and analyzed by a series of repeated measures, single-factor ANOVAs a separate analysis was performed for each comparison. The ANOVAs were calculated after each estimate was first subtracted from 100, allowing an assessment of whether estimates were significantly different from the initial estimates (100%). Sig- nificance levels were set atp .01 because four F-tests were performed on the data from each session. Threshold procedure. A forced-choice, modified, single-staircase procedure through five reversals was used to establish olfactory thresholds for the individual isomers of EE3M2H. A trial consisted of the presentation of two polypropylene bottles in rapid succession and in counterbalanced order. One bottle contained 10 ml of a given con- centration of the test odorant dissolved in light, white, mineral oil, and the other