J. Soc. Cosmet. Chem. 28 357-365 (1977)¸ 1977 Society of Cosmetic Chemists of Great Britain The influence of perfumes on the sensitising potential of cosmetic bases I. A technique for evaluating sensitising potential M. F. BRULOS*, J.P. GUILLOT% M. C. MARTINI* and J. COTTE* Received 4 January 1977 Synopsis A technique was developed for determining the sensitising potential of cosmetic products in the albino guinea-pig. It consists of the administration of Freund's complete adjuvant by intradermal injection and subsequent application of the test substance topically using an occlusive patch. The technique is therefore particularly well suited for the testing of finished products. INTRODUCTION At both national and European levels legislation concerning cosmetic products is now being drawn up. French legislation already demands a range of animal tests relating to ocular and cutaneous irritation (1) but as yet official requirements do not include sensitisation tests. The incidence of sensitisation reactions resulting from the use of cosmetic products is relatively low representing about four cases per million units sold (15). The performance of sensitisation tests on healthy human subjects is forbidden in France - such tests pose delicate ethical problems - it is therefore pertinent to search for a simple test to perform on animals. A review of the literature reveals that the techniques for such tests are diverse and numerous (6) their plethora being perhaps an indication of their limitations. All the test methods use a limited number of animals and rely upon increasing the sensitivity of the test animal in one or more of the following ways (7) (8): the use of high con- centrations (20) (3). This is rarely suitable for cosmetic bases as tests are frequently carried out on the undiluted product the special preparation of the treatment area (scarification or stripping) to accelerate penetration (18) the use of Freund's adjuvant (11) (12). Frequently a route of administration is used which is incompatible with the texture or consistency of most cosmetic products (4) (11) (12) (9). * Institut de Pharmacie Industrielle, Cosm6tologie, 8 avenue Rockefeller, 69008 Lyon, France. t Institut Francais de Recherches et Essais Biologiques, Les Oneins, B.P. 109, 69210 L'Arbresle, France. 357

358 M. F. Brulos et al. A test suitable for use with most cosmetic substances must meet the criteria out- lined below. Elimination of risks of parasitic irritation. Detection of weak sensitisers. Protocol design to suit finished products, i.e. sufficiently sensitive to function without increasing the concentration of the test product - topical rather than intradermal appli- cation of the test substance. Intradermally injected substances are sometimes absorbed with difficulty, particularly in the case of such cosmetic products as talcum powder and pigmented cosmetics, and may give false positive results since the dermal-epithelial barrier is bypassed. Evaluation of reaction site by a simple but stringent test using only clearly positive macroscopic readings backed up by histological examinations. Interpretation of response so that an 'all or nothing' result is obtained. A bibliographic study of those factors influencing the induction of release of a hyper- sensitive reaction, coupled with a number of preliminary trials (2) has led to the develop- ment of a technique which meets the above criteria. PRINCIPLE Sensitisation in the guinea-pig is induced by intradermal injections of Freund's adjuvant and by topical applications of the test substance under occlusive dressings. After a rest period of 12 days a single challenge application of the test substance, again under an occlusive dressing, provokes the appearance of a sensitisation reaction. The use of occlusive patches to increase the hydratation of the skin and permeability of the stratum corneum (10) plus the administration of Freund's complete adjuvant to maximize the immunological response gives the technique sufficient sensitivity to detect even weak allergens. EXPERIMENTAL PROCEDURE Animals Albino Hartley guinea-pigs of both sexes are used. They weigh between 300 and 400 g. ACCOMMODATION AND DIET Two weeks before the start of the study, and for the 40 days duration of the study the animals are kept in an air conditioned animal house in cages of five measuring 600 x 540 x 315 mm. The cages have grilled bases to eliminate soiling with faeces, litter, etc. Each animal is fed 50 g of granules per day (granules Cobaye U.A.R. N ø 114). This diet is supplemented with carrots. Water is freely available. SCREENING FOR PRIMARY IRRITATION Before commencing the sensitisation study it is essential to check that the test substance does not cause primary irritation (application of test substance for 48 h under occlusive patch using six guinea-pigs). All substances producing primary irritation are eliminated unless destined to be used in a diluted form. For these latter cases the minimum dilution which does not cause