316 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table IV. Claimed action No. Product class Biological assay Chemical assay Anti-wrinkle (for older 1 Face cream a a a* --•- -- women) 2 Face cream a a a -- -- 3 Throat cream a a a -- -- 4 Face cream a a a -- -- 5 Face cream a a a -- -- 6 Face cream a a a -- -- 7 Face cream a a a -- -- 8 Eye cream a a a -- -- 9 Face lotion a a a -- -- 10 Face cream a a a -- -- 11 Placenta cream a a a -- -- 12 Eye cream a a a -- -- 13 Night cream a a a -- -- 14 Night cream a a a -- -- 15 Face cream a a a -- -- 16 Face cream a a a -- -- 17 Placenta cream a a a -- -- 18 Face cream f b/d f + :1: 0.0025 •o DES 19 Placenta cream a a a -- -- 20 Throat cream a a a -- -- 21 D6collet6 cream a a a -- -- 22 Face cream a a a -- -- 23 Face cream a a a -- -- 24 Face cream a a a -- -- 25 Face cream a a a -- -- 26 Placenta cream a a a -- -- 27 Beautifying oil a a a -- -- 28 Face cream a a a -- -- 29 Face cream a a a -- -- 30 Royal yelly cream a a a -- -- 31 Placenta extract oil a a a -- -- 32 Idem aqueous a a a -- -- 33 Face cream a a a -- -- 34 Face mask a a a -- -- 35 Face cream a a a -- -- 36 Face cream a a a -- -- 37 Face cream a a a -- -- 38 Face cream a a a -- -- 39 Face cream a a a -- -- Development of the 40 Bust cream a a a -- -- female breast 41 Paper plaids a a a -- -- 42 Paper plaids a a a -- -- Depression of 43 Body cream f f f + 0.1 •o DES abnormal hairgrowth 44 Body cream-mild f f f + 0.01 • DES in the female 45 Body cream a a a -- -- * Cytological evaluation (see preparation and staining of cytological specimen). • -- ----Oestrogenic action absent + = oestrogenic activity present. • Results of quantitative biological assay see Table V.

OESTROGENIC SUBSTANCES IN COSMETICS 317 acetamide (BSA). Heat at 60øC for 10 min. Blow off excess BSA. Dissolve in 250 [tl carbon disulphide. Inject ca 3 [tl on the following. Glass column: 5•o SE-30 (or 5•o OV-17) on Chromosorb G. 1500 mm x 2 mm internal diameter. Temperature of column for SE-30: 190øC and for OV-17' 230øC. Flame ioniza- tion detection. Detector FID, 240øC. Carrier gas nitrogen' 30 ml/min. RESULTS AND DISCUSSION The results of biological and chemical assay for oestrogens of 45 market samples of cosmetic products, which have been purchased in the Netherlands in the second half of 1973 are tabulated in Table IV. Of thirty-nine anti-wrinkle preparations (including preparations containing placental extract) only one (18) contained detectable oestrogenic activity on investigation with the biological screening method. This finding was confirmed by the chemical methods.* From these data it can be concluded that oestrogenic substances were not present generally in anti-wrinkle cosmetics on the Dutch market. Sample 18 has been evaluated quantitatively with the quantitative biologi- cal method. (see Table V). The biological data (100-200 Mouse Units per g, which corresponds to 0.0010-0.0020•o oestrone equivalents) were in fair agree- ment with the chemical findings (0.0025•o DES), as the biological activity of DES, Table V. Quantitative biological assay of sample 18 Dosage per animal (~ mg sample) Cytology of vaginal smears Result 5* a/b f/d a/c 10 e/f e e + 10 d/e d/e e + 20 f f f/d + 40 e/f e/f e/f + lOO f/f f f + 0.075 Ilg oestrone e/f e/f e/f b/c c + 0.15 [tg oestrone f e f f f e + *Corresponds to 0.5 to 1 Mouse Unit (0.05--0.10 t•g oestrone). Conclusion: 1 g of sample 18 contains 100-200 Mouse Units (equivalent to 0.001--0.002 % oestrone). N.B. Sample 18 has been assayed chemically results 0.0025 •o diethyl- stilbestrol. * Some other hormonal, non-oestrogenic substances with presumed anti-wrinkle action, as ethisterone and pregnenolone, have been identified and determined in several samples. The analysis will however be described in a subsequent paper.