APPRAISAL OF METHODS FOR DETECTING PRIMARY SKIN IRRITANTS 751 been studied in biopsied skin samples. Alteration in the levels of the enzymes were related to changes in the steroid metabolism of the skin, the degree of keratinization and the dermal collagen synthesis respectively. An important disadvantage with the techniques used is that sufficient skin has to be biopsied to enable several biochemical assays to be performed using different substrates. A limitation of the hydroxysteroid dehydrogenase assay is that only those steroids with hydroxyl groups may be determined and if specific assessments are required only steroids with a single hydroxyl group can be used. The assessment makes no allowance for alterations in cutaneous steroid metabolism resulting from endocrine disturbances or from alterations in local concentrations due to the action of the circulatory system. The assay for arginase, using the method devised by Rossmiller and Hoekstra (109) and the assay for protocollagen proline hydroxylase also would appear, on available evidence, to be of limited application and to be an unreliable guide to irritation. Another enzyme which has been investigated as a possible guide to the irritancy of cosmetic materials, is saccharase (110). The test was based on the inhibition of the enzyme by some anionic detergents which were known to irritate the skin by damaging the keratin. It would seem, however, that the same physical properties of those detergents which are responsible for damaging the keratin, would also denature other proteins including the enzyme saccharase. The choice of an enzyme which is not normally present in the skin, would seem to make this test for cutaneous irritants of rather questionable validity. Whereas biochemical assays of enzyme levels in tissue samples seem to be an unreliable guide to tissue damage not visible with the naked eye, the histochemical demonstration of enzymes is likely to be of more value. Although few studies of this type have yet been reported, Reid and Jarrett (111) have shown that vitamin A treatment results in an increase in the level of lysosomal hydrolases in the stratum granulosum. Similar changes were noted in skin treated with the irritant-cetyl trimethylammonium bromide (112). In these studies, changes in enzyme levels were present before changes were identified histologically. Thus changes in enzyme levels appear to be more sensitive indicators of tissue damage than histological techniques and would appear to offer a considerable advantage over the in vitro assays referred to above. Keratin denaturatiot• When a substance irritates the skin, it may damage the epidermal keratin

752 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS of the stratum corneum and the stratum lucidum, or it may penetrate deeper into the epidermis, thereby bringing about cytotoxic changes (113). With the exception of a thin layer of surface debris and lipids secreted by the sebaceous glands, the keratin layer constitutes the outward line of the defence to the skin to potentially harmful substances. It is possible that information as to how a substance alters the keratin layer and the essential substances con- tained in it, may be of considerable value in detecting damage which is not visible to the naked eye. The horny layer of mammalian skin is composed mainly of the protein keratin, water and hygroscopic substances such as lipids, mucopolysacchar- ides, amino acids and sugars (114-117). Blank (114) maintained that for human skin to retain its supple nature it should contain at least 10}/o water. Although the removal of the hygroscopic substances apparently does not affect the ability of the skin to be rehydrated, the water is readily lost at low humidities and high temperature (118). An increase in the number of free sulphydryl groups in the keratin is evidence of denaturation resulting from a breakage of the disulphide link- ages in the amino acid cystinc (119, 120). Colorimetric methods have been used to determine these groups (119, 121) and the values used as an index of keratin denaturation (121, 122). Irritants such as detergents may free and elute essential amino acids or lipids from the keratin layer (123). To investigate such an effect, an apparatus consisting of a double-walled chamber is used. The suspected irritant dis- solved in a suitable solvent is placed in the chamber in contact with the skin stimulated with electrically operated teflon rollers for a test period of 15 min. At the end of the treatment, the fluid plus the amino acids and other substances eluted from the skin is analysed using standard techniques. Although the original technique was designed to examine the effect of detergents, it could be readily adapted to examine the effects of other irritants in a suitable solution. The test period could be varied according to the potency of the test substance. The removal of lipids from the epidermis has been studied using open type techniques (124). In one such study, animals were anaesthetized and dipped up to their necks in a test solution such as n-hexane and the removed lipids identified using thin layer chromatography. In a human study, the effects of some organic solvents on the scalp were examined using a simple washing procedure. The eluted lipids were characterized and quantified biochemically (125, 126). The design of these techniques appears to be relatively simple and enables them to be readily available for use hz vivo