APPRAISAL OF METHODS FOR DETECTING PRIMARY SKIN IRRITANTS 755 and serve as an early indication of damage. This view is supported by the observation that, in skin treated topically with carcinogens, antiseptics, some cosmetic chemicals and solvents, all of which are known to damage the tissues, oxygen uptake is depressed (132-134). Changes in the skin which might be recorded as a reduction in cutaneous respiration include alterations in the pH of the surface layer due to changes in the composition of the sebaceous secretion (132-135) and in the meta- bolism of the epidermal cells. Amersbach and his colleagues (133) used the Warburg apparatus in their investigation of respiratory changes in skin samples obtained by biopsy from patients treated topically with irritants. Skin samples were incubated with a suitable substrate and the oxygen uptake measured manometrically. Alternatively, samples of normal skin were incubated in the substrate which contained the compound under investigation. Changes in the respiratory rate shown in such an experiment would thus reflect the progressive alterations in the metabolism of the skin sample. Although potentially useful, the excessive variations which exist in the respiration of the human skin depending on race, sex, genetic type and environmental background would seem to present considerable difficulties in the interpretation of the results of such an investigation. Even in a single individual, the respiration of the skin would be expected to vary according to the part of the body from which the biopsies are taken (136). Furthermore, as Amersbach and his colleagues (133) pointed out, in the evaluation of the safety of cosmetic substances where tests are normally carried out using occlusive conditions, the respiration even in undamaged skin is likely to be lower than normal. Occlusive patches are known to cause increased sweat- ing, hyperthermia and inflammation (4). Therefore, unless the cutaneous damage caused by the test substance is sufficient to cause appreciable changes in the rate of oxygen uptake, the technique would appear to be insufficiently sensitive. Recent evidence that skin respiration studies are of doubtful value in the investigation of skin irritants, was given by Brown (137). Although this author believed that changes in respiratory activity might be indicative of alterations in carbohydrate metabolism (138-142), he found that the changes in respiratory activity were less than differences shown in patch tests. Thus, on available evidence, the Warburg technique for detecting changes in skin respiration would seem to be an unreliable way of detecting damage. Using the Cartesian diver method, no more success was achieved in

756 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS detecting differences in the respiration of normal and psoriatic skin (143). However, when succinate was added to the incubation medium which already contained glucose, pyruvate and oxaloacetate, respiration in the psoriatic skin was greater than in the normal skin. This succinate-enhanced respiratory activity was depressed if further pyruvate or oxaloacetate was added. It is possible that competition for the succinic dehydrogenase might account for this observation (144). Details such as these allow the specula- tion that some useful information concerning alterations in cutaneous metabolism might be gained by means of respiration studies if a variety of substrates is used. Unfortunately at present, insufficient information seems to be available as to the most suitable substrates, or on the inter- pretation of in vitro results in terms of damage caused. Also, bearing in mind the experience gained by Brown (137), that respiratory studies did not give results which were consistent with the irritancy of a substance as determined using patch tests, investigations of oxygen uptake would not appear to be helpful in detecting primary irritants. Function of skin appendages As a result of applying cosmetics to the skin there is a chance that the ducts of the glands opening on to the surface or into the hair follicles will be blocked, and the secretions of the glands unable to reach the skin surface. Also, if the constituents of the cosmetic pass into the gland ducts, the secretory cells of the glands may be exposed to damage. This type of damage might be manifest either by an alteration in the composition of the secretion or in a complete suppression of the activity of the gland. Some methods used to examine the distribution and functioning of sweat glands were described in a recent review by ¾erbov (145) with reference to a clinical condition known as hypohydrotic ectodermal dysplasia. A simple method for detecting functional sweat glands in the skin is the starch-iodine reaction, which was originally described by Wada and Takagaki (146). Starch grains in the presence of iodine react with sweat to give a black precipitate in the pores of the gland ducts. The tech- nique has been used to detect increased sweating in people given intradermal injections of adrenaline and acetylcholine. A more recent method, which appears to be at least as efficient, is based on the reaction between the constituents of sweat and o-phthalaldehyde (147). Using this reagent, sweat pores are marked out as black puncta which are not readily removed by washing or bleaching. Since the o-phthalaldehyde also gives a black