TESTING FOR LONG-TERM TOXICITY 833 Contrarily, carcinogens applied to the skin may cause epithelial tumors but not at the site of application. An example is N-nitroso- methylbutylamine which, when applied to the skin of the back, caused carcinomas of the eyelids and of the nasal cavity in mice (31). These diverse results illustrate that one must not merely inspect the test site for skin tumors. To be certain of the results, it is necessary to do com- plete necropsies of the experimental animals. ENZYME INDUCTION In view of the fact that certain materials which are present in many perfumery agents may act as enzyme inducers, thus influencing sub- stantially the response to carcinogens, this subject should be mentioned. In most animal species, enzymes which are located in the microsomal fraction of the cell can detoxify a large number ot• compounds not nor- mally present in the organism. Such systems have been studied exten- sively by pharmacologists because of their effects on drugs and because of the effects of drugs on each other (32). In addition to their capacity to detoxify drugs, these enzymes can convert a number of carcinogens such as polycyclic hydrocarbons, aromatic amines, or azo dyes to less ac- tive or inactive compounds such as their ring-hydroxylated derivatives. An important characteristic of these microsomal detoxification systems is that administration of the appropriate compounds, both in vivo or in vitro, can induce an increase in the activity of these systems. Among the types of compounds which have this property of enzyme induction are numerous polycyclic aromatic hydrocarbons, both carcino- genic and noncarcinogenic (33). Other inducers are phenothiazines and a sizable number of derivatives ($4), certain other drugs including phenobarbital, certain insecticides, numerous natural products such as safrole (35), some terpenes, as found in cedarwood (36, 37), and synthetic and naturally occurring ttavones (38). Terpenes, safrole, and flavones are of special interest since they are representative compounds which are present in small amounts in many perfumery and flavoring materials currently in use. In many experiments, it has been shown that such materials can lessen the action of carcinogens. An example is the decrease in number of skin tumors induced by benzo (a)pyrene in mice which were also treated with 5,6-benzottavone 09-naphthottavone) (38) (Table XI). The terpenes present in sweet orange oils inhibited considerably the activity ot• benzo(rst)pentaphene (dibenzopyrene) (39).

834 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table XI 5,6-Benzoflavone as Skin Tumor Inhibitor s Tumor Incidence Tumors per Primary treatment Carcinogen (%) Mouse Sesame oil b None 0 0 5,6-Benzofiavond, None 0 0 Sesame oil Benzopyrene c 40 0.7 5,6-Benzoflavone Benzopyrene c 17 0.3 a Data from Wattenberg and Leong (38). * 5,6-Benzofiavone, 6 mg/rnl of sesame oil, or sesame oil alone, was painted on the backs of 9-week old mice once daily for 3 days. c Two doses of 5 /•g of benzo(a)pyrene in acetone with 1% mineral oil were applied to the skin 24 hr after the last inducer or vehicle administration. COCARCINOGENS In contrast to the effects of compounds which can induce enzymes, thereby ultimately reducing the potency of many carcinogens, other nat- ural products have the ability to enhance or promote the action of car- cinogens. These so-called "cocarcinogens" generally have no carcino- genic activity. At most, they have an extremely weak effect (40). The best known of these agents are the phorbol esters, isolated from croton oil, from the seeds of Croton tiglium L. The structure of phorbol is shown in Fig. 3. In croton oil the phorbol is esterified by various fatty acids Cs (caprylic) to C•4 (lauric) (41). Other materials which have this capability are linalyl oleate and linalyl acetate (42), perfumery agents, and certain of the Tweens and Spans which have a lipophilic-hydrophilic structure similar to the phorbol esters. However, with the Tweens and Spans, extremely high doses are necessary to show any promoting effect (4-1). In some cases, irritant materials, when applied after a car- cinogen, had promoting action in skin carcinogenesis, but not con- sistently so. An illustration of the action of the phorbol ester fractions in enhanc- ing the effect of the strong carcinogen 7,12-dimethylbenz(a)anthracene CH20H OH • /•c% H$C •CcHH$ HSC / OH Figure 3. Structure of phorbol, parent compound of the phorbol esters