192 H.R. Watson et al. undesirable side effects such as an unpleasant taste, tingling and burning usually pre- dominate. Large numbers of compounds were examined in our study. When the method described above had become well established, a small panel of four to six subjects, chosen from the larger panel for their near average sensitivity was used for rapid initial screening of compounds. Topical Testing Like oral threshold determination, measurement of topical thresholds is complicated by the fact that individual sensitivities to cooling compounds differ considerably. Skin thresholds were measured as follows: 0.2 ml of a solution in petroleum ether (40-60 ø) was measured on to a circular area el0 cm •) of skin surface on the inside of the forearm, midway between the wrist and elbow. After 30 min, the subject was required to respond whether or not a cooling effect had been noticed during the period. CSome compounds were slow to act, but the main reason for the delay between dose and question was to allow the panelist to differentiate the chemical cooling effect from the inevitable but transient physical effect of the solvent evaporation.) Since degreasing the skin with solvent before application of the cooling compound did not change the threshold, and washing may have caused hydration, the skin was not treated before a threshold test. A panel of 50 subjects, tested by the above method, had thresholds for menthol of between 0.5 and 100 mg. As an example of the non-Gaussian grouping towards the sensitive end, the thresholds of 32 of the subjects lay between 2.0 and 10 mg, and only six subjects had thresholds of greater than 25 mg. It should be recalled that the oral threshold method was used extensively because it appeared to give the best measure of intrinsic cooling activity. Skin threshold levels were not of value for that purpose, and in addition they were confounded by the manner in which the compound deposited on the skin following evaporation of the solvent. Some compounds even crystallised, and therefore clearly gave spuriously high 'threshold' results. For these reasons, our topical testing did not rely on threshold methods. The aim of the topical testing was C a) to find, in general terms, those compounds which were most suitable for application in topical products and Cb) to match compounds with product types so that the overall effect of the addition of a particular cooling com- pound to a product could be judged. In view of the difficulties implicit in skin threshold testing, and the inevitable relationship between compound effectiveness and the medium in which it is applied, we concentrated on tests based on application in the product type. Thereby (b) above, for a particular composition, was satisfied directly, and Ca), the relative general characteristics of the compounds, gradually emerged as the number of tests in different media increased. This part of the study is on-going. The pattern of product oriented tests is exemplified by the following, which was used for aerosol shaving foams. The panel consisted of five men who were experienced in the effects of cooling compounds. The subjects applied approximately 0.5 g* of the shaving foam across the trigeminal/cheek area, wiped the area after 5 rain, and a trained observer recorded their comments during (normally) the first 10 min following application. The observer took * Unlike threshold testing, direct effectiveness testing is less influenced by the quantity of material applied. The concentration of the cooling compound in the medium is important, but compositions such as shave foams, which are applied in excess, are best tested in excess, it being implicit that they do not dry out during the period of observation.

New compounds with menthol cooling effects 193 particular note of time of onset, degree of cooling, and 'quality' (the meaning of the term 'quality' in this context is described below). The degree of cooling effect was scored on an 8-point scale (no effect, threshold, weak, weak-moderate, moderate, moderate-strong, strong, very strong). Repeat tests on different days were desirable a subject's repeat scores could vary by as much as 3 points on the scale. It was important that, as much as possible, the subject's skin was in the same state of hydration on repeat tests. (As an extreme example, the face is considerably more sensitive immediately following shaving than at other times of the day.) It was also desirable frequently to include as a control the base composition with no cooling com- pound this acted as a test on the panelists, and also permitted weighting of the scores since the base composition invariably caused the subjects to record sensations. Similarly, compositions of known very strong cooling effect were included as positive controls. SENSITIVITY OF DIFFERENT PARTS OF THE BODY Different parts of the body differ greatly in their sensitivity to the cooling compounds. No attempt has been made to determine the relative sensitivities in absolute terms, but the order of sensitivity is: eye tongue interior buccal region ano-genital area lip trigeminal area other face areas axilla inside forearm, breast other arm areas, thigh, back hands, feet palms,* soles. The eye is extremely sensitive, with thresholds probably measurable in nanograms, and to an extent that a simple test for cooling effect in a vol•tile compound is to hold the opened bottle near the eye. This test with menthol gives a sharp and very obvious sensation, which accords with consumers comments on the effect of mentholated shaving foam on the eye. Like menthol, synthetic cooling compounds also give a cooling sensation in the lungs (when added to cigarettes or inhaled as an aerosol spray) and to the gastrointestinal tract (when ingested). The order of sensitivity clearly follows a general order of increasing thickness of stratum corneum (26), and it seems probable that the sensitivity of an area is determined mainly by the ease with which the compound can penetrate this barrier. Further in- dication is provided by the fact that abraded or hydrated forearm skin has a reduced threshold compared to intact skin. Although the barrier role of the stratum corneum is probably dominant (27), it is likely also that the number of cold-sensitive nerve endings per unit area, and the efficiency with which the central nervous system processes nerve signals, vary with the location on the skin (3, 28). (During a search for an equipotency method based on mirror imaged body areas, the menthol thresholds of sixteen subjects were measured on the left and right forearm. Six were more sensitive on the right, and six on the left forearm. Four had no difference of threshold. There was no correlation with the side of the dominant hand of the subjects. Similar differences between left and right sides of the face have been noted in threshold tests. The study has not been taken further, but it seems possible that these differences are related to the efficiency of neural signal processing. At suprathreshold levels these bilateral differences are not detectable). * Some subjects have recorded a response when strong solutions are applied to the palms but our current view is that the effect is insufficiently distinct to be recorded positively.