NEW TRENDS IN COSMETIC EVALUATION 245 on cats and dogs were rather unsatisfactory. Better results were obtained on chinchilla rabbits, rats and suckling pigs. Very extensive studies are now being made on the penetration of vitamins and hormones through the skin. This type of research was practically im- possible for years because of the unavailability of the proper labeled chem- icals. Very recently, a few researchers have succeeded in tagging pre- viously unlabeled compounds and these can be easily incorporated into vitamins, male and female hormones and cortical extracts. These studies are time consuming and very expensive, but when completed they wilI answer the contentions of both the Food and Drug Administration and the cosmetic industry in an indisputable and recognized way. We believe that the use of radioisotopes is an important contribution to the methods the cosmetic industry has at its disposal to establish or improve the quality of its products. H^RM•.F. SSNESS In this section we originally planned to include bacteriological and toxi- cological investigations. However, as no important change has occurred in the approved methods for the bacteriological testing of cosmetics, since our paper entitled "Bacteriological and Dermatological Testing of Cos- roetics," given on December 3, 1947, was published in the •ourna/of ttw Society of Cosmetic Citemists (Vol. I, No. 3) we believe it best to refer the reader to it, and give more space to toxicological studies. ?xicological Met/wds. They are divided into two categories: those which investigate internal toxicity, and those which deal with external or local toxicity. Internal Toxicity. The internal toxicity of cosmetics can be tested three different ways. a. By intravenous i•0ection, whereby the extract is injected directly into the blood stream of rabbits. b. By intraperitoneal injection, whereby the extract is injected into the abdominal cavity of mice or rats. c. By feeding the extract either in drinking water, or mixed with solid food. This study is conducted for a period of two weeks to a lifetime, which corresponds approximately to two and one-half years for rats and one and one-half years for mice. Very often, cosmetic manufacturers are anxious to know the minimum lethal dose of the constituents of their products. This can be determined by either of the three methods. It gives them a numerical figure from which they can compute the toxicity of any concentration of these chem- icals. Save for a few exceptions, such as caustic chemicals, for instance, the MLD by intravenous injection is always smaller than the MLD by intraperitoneal injection, and the latter smaller than the oral MLD. Whereas MLD calls for the death of all test animals, MLD 50 calls for

246 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS the death of only half of the test animals. Needless to say, MLD 50 is always smaller than MLD 100. External Toxicity. This includes irritation of the skin, scalp, eye and mucous membranes. Three methods are currently used, all of them satisfactory within their own scope: (a) The animal intradermal injection method. This is the quickest, most accurate and inexpensive screening method. It is used with advan- tage before starting more elaborate and expensive investigations. For this reason, and because of the fact that it obtained wide acceptance during the past few years, it deserves a complete description. The main feature of the method is the preparation and injection of sterile extracts. The extracts of cosmetics or their compounds are made in an autoclave at a pressure of 15 pounds for twenty minutes. As the best extracts are isotonic, the vehicle of choice is normal physiological saline solution, containing 0.85 per cent of sodium chloride in freshly distilled water. However, when it is known that the substance to be tested is in- soluble in normal physiological saline solution, it can be extracted in sterile U.S.P. olive oil. Experience based on several thousands of tests in- dicates that the concentration of the extract should be 5 per cent whenever such materials would normally be patch-tested in their natural state on human subjects. The amount of the sterile extract to be aseptically injected is usually 0.5 cc. It seldom varies. Only the concentration of the extract is sub- ject to changes. The injection is absolutely painless and the elimination of the injected solution starts immediately after the injection. The results are obtained twenty-four hours after the injection. They show reactions which can be divided into five categories, or the absence of irritation. As stated before, the main purpose of the animal intradermal single in- jection method is to obtain rapid screening results in the determination of the presence of primary irritants. However, if an injection of the same amount of the same cosmetic is made on the same animal ten to fifteen days after the first one, it will indicate whether the cosmetic is a cutaneous sensitizer or not. The method is especially useful when the first injection indicates the absence of primary irritants prior to conducting a patch test on human subjects. Such a patch test is definitely unnecessary when either of the two injections shows positive results. Advantages of the method: 1. It is simple: no method is easier to apply. Any biology technician can use it. For this reason it has replaced the obsolete method of animal patch tests. 2. It is accurate, so much so that its results are almost always confirmed by the prophetic patch test.