308 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The use of hormonal substances is however not without risk. The hazards depend much on the concentration levels of these pharmacologically very active compounds (7). Thus it is important to develop biological and chemical methods to detect the hormonal activity, to identify the active compounds and to determine the levels of concentration. In this paper the methods developed for the detection of oestrogenic substances will be discussed. The biological methods developed for detection of oestrogenic activity are valid for the female sex hormones (oestradiol-1713, oestrone and oestriol) and other steroidal oestrogens (such as ethinyl-oestradiol) as well as for the stilbene- derivatives (such as diethylstilbestrol (DES) and hexoestrol). Chemical methods, on the contrary, focus the analysis on characteristic chemical structures and thus per- mit identification of the biologically active compound in many cases. This paper describes biological methods for qualitative and quantitative detection of oestro- genic activity and chemical methods for the detection of oestradiol-17[3, oestrone, oestriol and DES. Methods for the biological and chemical detection of other hormones will be described in a subsequent paper. PROCEDURES Sampling The forty-five samples were purchased from the Dutch market in the second half of 1973. Since no formula is stated on the labels or on the information pamphlets, the selection of the products to be examined was based exclusively on their beautifying or cosmetic claims such as: anti-wrinkle creams for the neck and around the eyes and sometimes for the face, particularly indicated for the older woman, with visible results after about 2 weeks of daily application: growth promoting effect on the female breast depression of abnormal hair growth in the female, such as hirsutism. Prices of the samples varied from $3 to $40 per unit. BIOLOGICAL METHODS The Allen-Doisy test (10) was chosen for use with cosmetics because of its specificity. Samples could in principle be administered orally or parenterally (subcutaneous, intravaginal, percutaneous, etc.). In the case of cosmetics, the advantage of percutaneous administration of the specimen without extraction is obvious however, for a quantitative assay it was necessary to extract the material to be examined and to administer the extract subcutaneously. The percutaneous method proved to be very useful in our hands for screening pur- poses, as it is simple, sensitive and rapid.

OESTROGENIC SUBSTANCES IN COSMETICS 309 Test animals Female mice--weight about 20 g--were castrated bilaterally. After 1 week the wounds should have healed and heat phenomena should have disappeared (control with vaginal smear, see below). This was followed by activation with 0.1 ml of 10 [tg oestradiol-1713/ml oil solution subcutaneously on three consecutive days the first sign of oestrogenic action should have appeared about 48 h after the first injection (control by vaginal smear). Animals which did not respond were discarded. This activation was repeated if the animals remained 2 weeks in di- oestrus a lower concentration of oestradiol-1713 (0.5 [tg/ml oil) was used in this case. Reactivation was practised in animals which had not been used for testing during the 2 weeks, as well as in animals which had a negative test result. One week after (re)activation the vaginal smear was checked. Immediately after this check (which should be negative), the animals can be used for the actual test. Mice were discarded if they weighed over 35 g. Administration of samples For screening, the cosmetic product was applied without extraction, on the shaven skin behind the head (the only place that cannot be scratched or licked by the animal). The samples were applied three times, viz. on the first day at 15.00 and on the second day at 10.00 and 15.00. The sample was applied by brush, which was for one sample only. The application was done as reproducibly as possible, and some control of the quantity applied was possible by backweighing. The mice (three per sample) were housed individually in glass jars with a minimum of sawdust. Although the percutaneous test cannot give exact quantitative results, its excellent sensitivity and its simplicity make it the method of choice for screen- ing. Extraction of the sample was only practised when a quantitative test was necessary 1 g of the sample was extracted with 50 ml peroxidefree diethylether. The aqueous layer was discarded. The ether was evaporated and the residue was dissolved in 10 ml 96•o ethanol 0.05, 0.2 and 1.0 ml of this solution were added to 1.5 ml of olive oil. Acetone was added to obtain clear solutions, after which the volatiles were removed under a stream of nitrogen. The remaining oil solution was injected (0.1 ml per animal per injection) at 15.00 (day 1) and at 10.00 and 15.00 (day 2), using 3 animals per concentration. (This was the same scheme as used in the case of percutaneous administration.) Three animals receiving the same treat- ment could be housed in one cage. Preparation and staining of the cytological specimen Vaginal smears were taken by means of a platinum or chromium loop which was adapted to the size of the vaginal opening. The loop was passed through a flame and dipped in distilled water. A droplet of water was placed on an object