j. Soc. Cosmet. Chem., 28, 53-64 (February 1977) Screening of fragrance materials for allergenicity in the guinea pig I. Comparison of four testing methods G. KLECAK, H. GELEICK, and J. R. F1LEY. * Pharmaceutical Research Department, F. Hoffmann-La-Roche & Co. Ltd., CH 4002 Basel, Switzerland. Received November I O, ] 975 •::: Synopsis ::':" An OPEN EPICUTANEOUS TEST (OET) is proposed for the detection of SKIN IRRITATION and CONTACT HYPERSENSITIVITY induced in GUINEA PIGS. Thirty-two compounds described in the :::: " literature as being ALLERGENIC for man were tested in the guinea pig by the OET technique, and for the ':i!' : purpose of comparison, by three other techniques, namely the DRAIZE TEST (DT), the MAXIMIZA- : ß • TION TEST (MT) and a test with FKEUND'S COMPLETE ADJUVANT (FCAT). In the OET, a high degree of correlation was found between the allergenicity of the tested compounds for the guinea pig and for :: :•:i• man. :.. : ß I. INTRODUCTION A considerable amount of work has been done over many years developing pro- cedures for detecting skin-irritating and/or sensitizing effects of chemicals on labora- tory animals, in order to preselect compounds likely to be well tolerated by man. As in general toxicology and pharmacology, the results of such tests on animals cannot be fully valid for humans because of interspecies differences in the absorption, me- tabolism, and excretion of the compounds concerned. Furthermore, in the case of topical medication and of cosmetics, the conditions of exposrtre, such as concentration, frequency of application, and site of contact, can never be identical in human use and in animal experiments. The problem of identifying contact allergens, in humans as well as in laboratory ani- mals, was first approached critically and on a scientific basis by Kligman (1, 2, 3) and by Magnusson and Kligman (4, 5). These authors carried out comparative tests of nu- merous drugs, industrial contactants and cosmetics with well-known sensitizing properties, by various so-called predictive procedures commonly used on humans (6- 21), and have shown conclusively that these procedures often fail to identify even known sensitizers. These procedures are, therefore, also likely to be inadequate for the recognition of the allergenicity of new synthetic compounds. By carefully analyzing all .•:

54 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS the factors influencing contact sensitization in man and in the guinea pig, and by includ- ing all these factors in the same experiment, these authors developed a testing tech- nique which they called the maximization test (MT). With this technique, they found a close correlation between clinical experience and experimental results. The authors conclude that by testing compounds on laboratory animals under extreme experi- mental conditions it should be possible to detect the whole sensitizing potentiality of a compound. This statement should permit the assumption that the probability of a compound caus- ing sensitization in humans could be estimated. The authors emphasize, however, that it is impossible, on the basis of animal experiments alone, to formulate fully reliable predictions valid for humans. The purpose of our work, taking into account the knowledge acquired by Kligman and Magnusson, was to develop a testing procedure for guinea pigs convenient for testing new synthetic chemical compounds intended for use in fragrances and cosmetics. Spe- cial attention was paid to developing a test which would be simple to perform, would simulate the conditions in human use, would yield quantitative data, and would mini- mize the effects of subjective factors in evaluating the results. Like Kligman, we checked the reliability of our test by performing concurrently 3 other tests commonly used in this field. We tested a series of compounds with a well-known sensitizing ca- pacity for man, in order to establish whether or not a correlation exists between clinical experience and the results of our animal experiments. For the first step in these investigations, we chose 32 compounds described in the literature as being allergenic for man. These compounds were tested concurrently, on groups of 6 to 8 guinea pigs each, by the open epicutaneous test (OET), the Draize test (DT), the maximization test (MT) and an intradermal test with Freund's complete adjuvant (FCAT), all described below. II. MATERIALS AND METHODS CHEMICAL COMPOUNDS Thirty-two compounds used in the perfume industry were tested. All these compounds were tested under code numbers, their nature and potential allergenicity being un--. known to the experimenters prior to testing. :, : ANIMALS The animals used were male and female outbred Himalayan white-spotted guinea pigs ß bred at the Institute of Biomedi'kal Research, Fiillinsdorf, Switzerland. The guinea pigs weighed 400 to 500 g. They were fed on pelleted feed supplemented with green: vegetables, carrots and vitamin C in the drinking water, all available adlibitum. TESTING METHODS OET.' All the compounds were tested undiluted as well as dissolved in acetone, ethanol, diethyl phthalate, etc., at concentrations of 30, 10, 3, 1, 0.3, 0.1, and 0.03 per cent (or less when necessary) in order to establish a dose-response curve making it possible to determine the minimal irritating and the maximal tolerated concentrations on an "all or