Participaciones de los usuarios en los foros del grupo 140

Cain and Cometto-Muniz have developed a standardised method for odor detection, nasal pungency and eye irritation threshold in humans of individual VOC as well as mixtures of VOCs.^'^'^^^

Cain and Cometto-Muniz measured nasal pungency thresholds in persons lacking a functional sense of smell, viz. anosmies, for whom odor did not interfere. Odor thresholds, on the other hand, were measured on subjects with normal olfaction, viz. normosmics, matched to the anosmies by age, gender, and smoking status, which are variables known to affect chemosensory sensitivity.

Cometto-Muniz and Cain have designed a two-altemative forced-choice procedure with presentation of increasing concentrations served to measure all chemosensory thresholds. The method requires participants to use the assigned nostril to select on each trial, the stronger of the two stimuli: one is always a blank, solvent, and the other a dilution step of the tested substance, starting with a concentration clearly below threshold. Selection of the blank triggers, on the following trial, presentation of the next step, i.e. a higher concentration, paired with a blank. Selection of the stimulus triggers, on the following trial, presentation of the same dilution step from a duplicate set, also paired with a blank. The procedure continues until the subject selects the substance over the blank five times in a row. The dilution step where this first occurs is taken as the threshold. Once the thresholds is measured for one nostril or eye, the other nostril or eye is tested. After this, testing begins with another stimulus. If the experimenter were testing localisation on the right nostril, he/she would be asked which presentation led to a stronger sensation in the right nostril. If the experimenter were testing localisation on the left nostril, he/she would be asked which presentation led to a stronger sensation in the left nostril. Next for all types of sensory endpoints, steps of increasing concentration will continue to be presented four times, paired with blanks, until the subject achieves 100 % detection at two successive concentrations. This is taken as the detection threshold.

Stimuli are delivered from cylindrical, squeezable, high-density polyethylene bottles (270-cm^ capacity) containing 30 cm^ of solution, see Figure 2.5. For measurement of odor and nasal pungency, the bottle closure has a pop-up spout that

fitted into the nostril being tested. Each nostril is tested separately. For measurements of eye irritation, the bottle caps held a tube that led a 25 cm^ measuring chamber, the rim of which is placed around the eye. Each eye is tested separately. A squeeze of the bottle delivered a puff of vapor into the measuring chamber where the eye was exposed. A polyethelene dust cover closed the open end of the measuring chamber when the bottle is not in use. Cometto-Muniz and co-workers have recently devised a new stimulus delivery system, or glass vessel, and used it to measure nasal pungency threshold in anosmies. The design of the 270-cm^ glass vessels aimed at producing environmentally realistic thresholds through the following improvement of the squeeze bottles: (i) the avoidance of dilution of the stimulus by providing a tight nose-piece-nostril connection; (ii) increase in the volume of stimulus vapour available to accommodate a human sniff completely; and (iii) elimination of any low-odor background inherent in the plastic squeeze bottles. The results showed that nasal pungency thresholds measured via the glass vessels were significantly lower than those measured via the squeeze bottles while showing the same trend within the members of each series.

Quantification of all vapour stimuli, whether in the squeeze bottles or in glass vessels, is achieved through gas chromatography (FID detector) by direct sampling via a gas sampling valve or a gas-tight syringe. Concentration measurements are made off­ line right after preparation of the stimuli, concomitantly with testing and after all subjects had been tested, to confirm stability. All readings are then referred to those of the undiluted chemical, assumed to represent saturated vapour at room temperature, 296K.

In addition to measurement of threshold values for single VOC, Cometto-Muniz determined dose-response, i.e. detectability, functions for binary mixtures via the two- altemative forced-choice procedure with an ascending concentration order of presentation.^"^’^^ To obtain the stimulus-response, i.e. psychometric, functions for individual compounds, a series of twofold dilution steps of the individual compounds, a series of twofold dilution steps of the undiluted chemical (100% v/v) is prepared, i.e. 50,25,12.5,6.25, etc. % v/v. If two chemicals A and B have different chemosensory potency, their detectability functions will be displaced along the concentration axis with the function for the more potent chemical displaced to the left.

Figure 2.5. Monorhinic nasal testing via pop-up spout on the cap of the squeeze bottle.

Having these functions, for any concentration of one chemical, one can interpolate the corresponding probability of detection (p) for that concentration. With this value of “p” an interpolation in the function for the other chemical can be done in order to obtain the sensory equivalent concentration of each chemical. In summary, Cometto-muniz and co-workers found the concentrations of this second chemical, e.g. p = 0.60. These two concentrations were called sensory equivalent. This procedure allows the scientist to express the mixture of a certain concentration of chemical A with a certain concentration of chemical B in terms of concentration of only one chemical, A or B. For instance, the concentration of chemical B into can be transformed into the sensory equivalent concentration of chemical A. Then, the two concentrations are added for

each of the various mixtures and compare their trends in detectability with the corresponding trend for detectability of the single chemical.

2 .2 .1 . C h em osen sory d etecto b ility o f single chem ical

Detectability functions for single compounds across a wide and orderly array of substances, i.e. homologous series can provide insight into the functional properties of each sensory modality investigated. They can also provide an additional way to probe the role of physicochemical properties, arranged as a continuum along homologous series, on the sensory potency of chemicals. Odor thresholds are always below, most times well below, nasal pungency and eye irritation thresholds. The investigations have also shown that all sensory thresholds tend to decline with carbon chain length in homologous series. Quite often, though, and particularly for the three to five members, odor thresholds decline faster than nasal pungency thresholds with carbon chain length. Therefore the gap between odor and pungency grows larger across those first few members of each series. Across all the series studied, the size of the gap has varied between one and five orders of magnitude. The decline in odor thresholds has tended to approach a plateau whereas the decline in nasal pungency thresholds has tended to show an abrupt cut-off. From certain homologue on, e.g. octan-l-ol, octyl acetate and propyl benzene, a threshold for nasal pungency failed to be reached, even with presentation of undiluted chemical. As stressed by Cometto-Muniz and Cain, substances not usually regarded as irritants, e.g. heptan-l-ol, nonan-2-one, not only can be detected by anosmies, i.e. they have pungency, but their pungency threshold is lower than that of more typical irritants, i.e. methanol, acetone.^'^^

Eye irritation thresholds, as a rule, fell into the register with nasal pungency thresholds. Nevertheless, for some of the homologous series studied, the highest member, i.e. longer chain-length, could not evoke nasal pungency but did evoke eye irritation. This might indicate broader chemesthetic responsiveness to airborne chemicals in the ocular mucosa than in the nasal mucosa.^'^^

Nasal localisation, i.e. determination of whether a vapor has entered the right nostril or the left, has provided a way to measure chemesthetic potency in normosmic persons. Nasal localisation thresholds for homologous n-alcohols and for selected terpenes tended to agree between normosmic and anosmic. In turn, these thresholds were either the same or only slightly higher, typically less than half an order of

magnitude, than the respective nasal pungency thresholds, but were as expected substantially higher than the respective odor thresholds.^'^^

Both the nasal thresholds (pungency and odor) for the initial set of 42 VOCs were plotted as a function of a simple physicochemical property, e.g. saturated vapor concentration at room temperature, 296K. Pungency thresholds taken as a whole exhibit a linear relationship with saturated vapour, r = 0.97, having a slope of 1.02. Pungency for individual homologous series conforms to the general picture (slopes: 0.90 for the alcohols, 1.07 for the acetates, 1.06 for the ketones, 1.17 for the alkylbenzenes, and 0.95 for miscellaneous compounds. The linear correlation with slope close to 1.00 suggests that when a certain uniform percentage of vapour saturation is achieved, nasal pungency would occur in the anosmies if it were to be evoked at all.^'^’^'^^

On the other hand, other thresholds as a whole depicted a more substance-to- substance scatter than pungency thresholds. They generally failed to show a linear relationship with saturated vapour. The odour for no individual homologous series exhibited a strong a correlation with vapour saturation as did the pungency thresholds for all series and miscellaneous substances grouped together. For odor, the best correlation occurred with the ketones, where r = 0.95, and the worst for acetates, r = 0.87. The slopes of the relationships for odor thresholds commonly departed from unity. 1,2,3,5-11

2 .2 .2 . C h em osen sory detecta b ility o f m ixtures

The chemosensory discomfort experienced in any place may in principle come from a single chemical compound but more commonly comes from a mixture. Hence, in order to understand, and predict sensory impact, both single compounds and mixtures require study. The level of understanding of the impact of single compounds has reached a state of maturity that quite naturally invites attention to mixtures, though at this point only simple ones.

Odor responses, at suprathreshold level, to very simple mixtures of volatile compounds have been the subject of a number of investigations on human olfaction.^^'^^ The typical outcome has shown that the intensity of a mixture is significantly less than the sum of the intensities of the components. The particular combinatorial rules to predict odor intensity of mixtures have proven straightforward in the binary case. Hence from knowledge of the perceived intensities of unmixed components, the perceived

intensity of a mixture can be calculated with reasonable precision. At suprathreshold level, odors seem to antagonise one another, apparently more as they become stronger. One might imagine that antagonism would therefore diminish toward threshold and this seems roughly to be so.

Comparatively few studies have explored odor perception of chemical mixtures at threshold level. The data generally suggest that a mixture reaches its odor threshold when each of its components lies below its individual threshold, a result that indicates some degree of agonism among components.^®’^ In fact, such odor agonism at threshold is usually larger than seen at suprathreshold level, a result in line with the observation that weak odors add more potently than stronger o d o r s . C a i n and co-workers have also seen partial agonism among odorants for the perception of odor thresholds in mixtures of up to nine components.^^

Chemesthesic responses, at suprathreshold level, to mixtures of substances, have received attention from studies done principally on rodents^^'^^ and to a much lesser extent, on h u m a n s . T h e studies on rodents have employed the classical respiratory depression technique developed by Alarie that measures Results have shown cases of competitive agonism^^"^^ or antagonism^^ depending on the particular mixtures studied. The studies on humans have indicated, that in contrast to the outcome for odor, the suprathreshold nasal pungency evoked by formaldehyde and ammonia adds, or even, potentiates to produce the pungency of the binary mixture.

Regarding chemesthetic responses at threshold level for the respiratory depression technique, the use of an RDo value, an extrapolated threshold concentration for the respiration effect has been s u g g e s t e d . ^ T h i s term was introduced for compounds with a low slope of the log concentration-response curve, and which, even at high concentrations, were not able to produce a 50% decrease in respiratory rate.^^ Pharmacologically, such compounds are considered partial agonists of the sensory irritation receptor, having a low intrinsic activity. It is interesting to speculate whether the decrease in intrinsic activity seen along some homologous^^ series reveals itself as a change on the quality of the threshold nasal pungency response of anosmies, that changes from a sharp, crisp sensation for lower homologous to a ‘pastel’ sensation for higher homologs.^

Cometto-Muniz and Cain measured sensory thresholds: odor, nasal pungency and eye irritation for individual VOCs (propan-l-ol, hexan-l-ol, ethyl acetate, heptyl acetate, pentan-2-one, heptan-2-one, toluene, ethyl benzene and propyl benzene) and

mixtures of them.^^ Various degree of stimulus agonism effects were observed for each of the three sensory channels when testing mixtures. As the number of components and the lipophilicity of such components in the mixtures increased, so did the degree of agonism. Synergistic stimulus agonism characterised the eye-irritation response for the most complex and the most lipophilic mixtures.

Studies of the functional properties of the olfactory and trigeminal chemosensory systems in humans have often focused on threshold measurements on a single point on a dose-response, i.e. detectability, function or on suprathreshold functions over a range of concentration. Odor and chemesthetic detectability functions provide a means to compare olfactory and trigeminal functionality as both chemosensory systems cross the boundary between threshold and suprathreshold responses. Using butan-l-ol and heptan-2-one and more recently butyl acetate and toluene, Cometto-Muniz et a l measured detectability functions for the odor, nasal pungency, and eye irritation of these four substances alone and in binary mixtures (butan-l-ol / heptan-2-one or butyl acetate / t o l u e n e ) . W h e n all stimuli, single and mixtures, were transformed into concentration units of one, or the other chemical, a single function could fit at all data from the same sensory end point with a correlation of 0.91 or higher. The outcome lends supports to the notion of chemosensory agonism, in the sense of dose additivity, between the members of binary mixtures presented at peri threshold levels.

2.3. Quantitative structure activity relationships for chemosensory