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1385. Croton oil: The siamese twins at it again
CANCER RESEARCH
593
In 1959, the US Food Protection Committee described a "practical procedure for evaluating carcinogenicity". This included continuous feeding of various levels of the test substance to rats and mice for their normal lifespans and to dogs for at least 4 yr; repeated topical application of different doses to the skin of mice for the normal lifespan; and a single subcutaneous injection in mice of not more than 100 mg of'the test sub.stance with observation of the animals until death. It seems highly doubtful whether all this information is available on any food additive. To see whether the information derived from the last two tests would afford additional evidence of carcinogenic potency or lack of it, six compounds on which long-term feeding studies had been conducted were selected, three of them carcinogens or suspect carcinogens [aminotriazole, Flectol H (a polymer of 1,2-dihydro-2,2-4-trimethylquinoline) and Aramite (2-(p-tert-butylphenoxy)isopropyl 2-chloroethyl sulphite)] and three non-carcinogens (butylated hydroxyanisole, dioctyl adipate and methoxychlor). In the subcutaneous test, single injections of 10 mg/mouse were given and in the skin application test, 0-I or 10 mg of each test compound was given weekly. In addition to having no adverse effect on survival, none of these compounds produced tumours at the site of subcutaneous or dermal administration. It is concluded that at least m the case of compounds with low carcinogenic potential, these two procedures cannot replace tests employing other routes of administration, and also that they are extremely unlikely to provide significant additional information. [Whilst we fully agree with the authors' inference that carcinogenic activity is best assessed by the oral route, we attach more value than they do to other routes of administration. In the case of polycyclic aromatic hydrocarbons, for example, carcinogenicity would have gone undetected had just the oral route been used. Until more is known therefore about the nature of the carcinogenic process it would be unwise to rely solely on one route of administration. As far as the UK is concerned the authorities recommend that in the testing of food additives for carcinogenicity both the oral route and wherever possible a parenteral route should be employed.] 1385. Croton off: The siamese twins at it again Hecker, E., lmmich, H., Bresch, H. & Schairer, H. U. (1966). 13ber die Wirkstoffe des Croton/51s. VI. Entziindungsteste am M~iuseohr. Z. Krebsforsch. 68, 366. Van Duuren, B. L., Langseth, L., Sivak, A. & Orris, L. (1966). The tumor-enhancing principles of Croton tiglium L. II. A comparative study. Cancer Res. 26, 1729. Gallantly, the schools of Hecker and Van Duuren soldier on in their determined efforts to identify the biologically-active principles of croton oil (I). Some measure of success was achieved with the discovery that phorbol myristate acetate is possibly one of the active constituents (Cited in F.C.T. 1966, 4, 525). Hecker et al. (cited above) now suggest that measurement of the inflammatory activity of the various fractions of I would serve as a reliable index of their potential co-carcinogenicity. A rapid quantitative assay of inflammatory activity (confirmed histologically) has been developed following the application of irritant components of I to the mouse ear. Values (#g/ear) for the EDso (dose required to produce inflammation in 50 ~o of the animals) of I, AI and phorbol triacetate [similar in toxicity and pharmacological activity to croton seed (II)] were 0-5-1.2, 0.01 and 1-2-3.8, respectively. However, phorbol and alcohol-phorbol, which have also been isolated from I, had very low inflammatory activity. Van Duuren et al. (cited above) have compared the tumour-promoting potential of I and its trace components A and C, preliminary findings on which have been reported earlier (Cited in F.C.T. 1965, 3, 341). Using 7,12-dimethylbenz[a]anthracene as a tumour initiator in mice, A and C were found to be five times more potent than H as tumour promoters. A few applications of as little as 5 #g A or C readily produced papillomas with a latent period
594
CANCER RESEARCH
of about 30 days. Very few of these tumours regressed and when these three promoters were tested alone for carcinogenicity, turnout incidence was very low indeed. Skin damage caused by II and fraction C was also found to be very much milder than that caused by I. Following a single application of II (25 #g) or fraction C (5 #g) to the skin of mice, inflammatory reactions were detected histologically within 4 hr, becoming maximal at 24 hr and subsiding thereafter. Diffuse epidermal hyperplasia was also noted after 24 hr and this was the only skin reaction observed after chronic exposure to II or fraction C. From the findings of these authors and other workers it appears that skin damage due to I, II or their fractions is not necessarily related to the tumorigenic or promoting effect of the material. Emphasis was placed on the importance of chemical specification in determining the biological activity of different batches of I. Of particular interest was the detection of alkylating agents (possibly including an epoxide) in I which may well account for the alleged carcinogenicity of certain samples of 1.
593
In 1959, the US Food Protection Committee described a "practical procedure for evaluating carcinogenicity". This included continuous feeding of various levels of the test substance to rats and mice for their normal lifespans and to dogs for at least 4 yr; repeated topical application of different doses to the skin of mice for the normal lifespan; and a single subcutaneous injection in mice of not more than 100 mg of'the test sub.stance with observation of the animals until death. It seems highly doubtful whether all this information is available on any food additive. To see whether the information derived from the last two tests would afford additional evidence of carcinogenic potency or lack of it, six compounds on which long-term feeding studies had been conducted were selected, three of them carcinogens or suspect carcinogens [aminotriazole, Flectol H (a polymer of 1,2-dihydro-2,2-4-trimethylquinoline) and Aramite (2-(p-tert-butylphenoxy)isopropyl 2-chloroethyl sulphite)] and three non-carcinogens (butylated hydroxyanisole, dioctyl adipate and methoxychlor). In the subcutaneous test, single injections of 10 mg/mouse were given and in the skin application test, 0-I or 10 mg of each test compound was given weekly. In addition to having no adverse effect on survival, none of these compounds produced tumours at the site of subcutaneous or dermal administration. It is concluded that at least m the case of compounds with low carcinogenic potential, these two procedures cannot replace tests employing other routes of administration, and also that they are extremely unlikely to provide significant additional information. [Whilst we fully agree with the authors' inference that carcinogenic activity is best assessed by the oral route, we attach more value than they do to other routes of administration. In the case of polycyclic aromatic hydrocarbons, for example, carcinogenicity would have gone undetected had just the oral route been used. Until more is known therefore about the nature of the carcinogenic process it would be unwise to rely solely on one route of administration. As far as the UK is concerned the authorities recommend that in the testing of food additives for carcinogenicity both the oral route and wherever possible a parenteral route should be employed.] 1385. Croton off: The siamese twins at it again Hecker, E., lmmich, H., Bresch, H. & Schairer, H. U. (1966). 13ber die Wirkstoffe des Croton/51s. VI. Entziindungsteste am M~iuseohr. Z. Krebsforsch. 68, 366. Van Duuren, B. L., Langseth, L., Sivak, A. & Orris, L. (1966). The tumor-enhancing principles of Croton tiglium L. II. A comparative study. Cancer Res. 26, 1729. Gallantly, the schools of Hecker and Van Duuren soldier on in their determined efforts to identify the biologically-active principles of croton oil (I). Some measure of success was achieved with the discovery that phorbol myristate acetate is possibly one of the active constituents (Cited in F.C.T. 1966, 4, 525). Hecker et al. (cited above) now suggest that measurement of the inflammatory activity of the various fractions of I would serve as a reliable index of their potential co-carcinogenicity. A rapid quantitative assay of inflammatory activity (confirmed histologically) has been developed following the application of irritant components of I to the mouse ear. Values (#g/ear) for the EDso (dose required to produce inflammation in 50 ~o of the animals) of I, AI and phorbol triacetate [similar in toxicity and pharmacological activity to croton seed (II)] were 0-5-1.2, 0.01 and 1-2-3.8, respectively. However, phorbol and alcohol-phorbol, which have also been isolated from I, had very low inflammatory activity. Van Duuren et al. (cited above) have compared the tumour-promoting potential of I and its trace components A and C, preliminary findings on which have been reported earlier (Cited in F.C.T. 1965, 3, 341). Using 7,12-dimethylbenz[a]anthracene as a tumour initiator in mice, A and C were found to be five times more potent than H as tumour promoters. A few applications of as little as 5 #g A or C readily produced papillomas with a latent period
594
CANCER RESEARCH
of about 30 days. Very few of these tumours regressed and when these three promoters were tested alone for carcinogenicity, turnout incidence was very low indeed. Skin damage caused by II and fraction C was also found to be very much milder than that caused by I. Following a single application of II (25 #g) or fraction C (5 #g) to the skin of mice, inflammatory reactions were detected histologically within 4 hr, becoming maximal at 24 hr and subsiding thereafter. Diffuse epidermal hyperplasia was also noted after 24 hr and this was the only skin reaction observed after chronic exposure to II or fraction C. From the findings of these authors and other workers it appears that skin damage due to I, II or their fractions is not necessarily related to the tumorigenic or promoting effect of the material. Emphasis was placed on the importance of chemical specification in determining the biological activity of different batches of I. Of particular interest was the detection of alkylating agents (possibly including an epoxide) in I which may well account for the alleged carcinogenicity of certain samples of 1.