THE TOXICOLOGY OF ARTIFICIAL COLOURING MATERIALS 427 aminoazo compounds, derivatives of phenylazo-2-naphthol and other azo compounds containing neither amino nor hydroxyl groups. Of primary interest here is the group of colourings, which includes such compounds as Carmoisine and Sunset Yellow FCF, which are derivatives of phenyl- or naphthylazo-2-naphthol. Despite a few suggestive earlier observations, the weight of evidence today leads to the conclusion that food colourings of this class are not carcinogens. Liver carcinogenesis by fat-soluble colours, of which p-dimethyl- aminoazobenzene is the prototype, and by water-soluble colours such as Ponceau 3R (26,27) creates considerable problems. Closely-related materials, for instance Ponceau 2R, are suspect even if liver tumours are reported to have been produced by analogues of this sort, one cannot be certain that adequate steps were taken to exclude traces of carcinogen (i.e. Ponceau 3R) that might have been present as impurities in the product tested. More fundamental, and more controversial, is the question - are low levels of such hepatocarcinogens acceptable for external topical applica- tion? Local carcinogenesis elicited by application to skin or mucous membranes is, of course, of special interest. Fortunately the colouring materials with which we are concerned do not produce such effects nor has carcinogenesis, or promoting action being attributed to any of the colourings employed in food or cosmetics. Bladder carcinogenesis has been studied (25) but has merely confirmed other observations, such as the carcinogenicity of Ponceau 3R and the non-carcinogenicity of Ponceau 2R, Blue VRS and Patent Blue V (28). Weighing up the evidence, it seems reasonable to accept the use of colourings that are hepatotoxic or hepatocarcinogenic in animals, provided that use is severely restricted to preparations for external application where ingestion will not occur. Tests by subcutaneous injection For some unaccountable reason, synthetic colouring matehals have been extensively tested for carcinogenic potential by repeated injection under the skin of rats. Of those colourings that have been investigated in this way, a substantial proportion have produced malignant tumours, usually fibrosarcomas, at the site of injection. It is likely that many, if not most, of the colourings not yet tested in this way are capable of inducing subcutaneous sarcomas. The extensive background to this problem has been reviewed in con- siderable detail by Grasso and Golberg (29). When the host of complicating

428 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS factors has been taken into account it becomes clear that the production of local sarcomas by injected colourings is the indirect result of the peculiar circumstances of the test procedure. The colourings themselves, by causing tissue damage, create local lesions which are not in any way connected with carcinogenicity (30). From certain types of lesion sarcomas evolve, but this is a secondary and non-specific outcome only indirectly related to the nature of the colouring injected. Thus the resulting sarcoma provides no evidence, for or against carcinogenicity of the test material. An analogy might be useful. There is the striking instance of the relationship between the production of oxalate bladder stones and bladder tumours in the rat. Ethylene glycol and polyoxyethylene-8-stearate (given at absurdly high dietary levels) have been labelled as carcinogens because they give rise to oxalate calculi in the rat bladder, and because - as a result of the presence of the calculi - turnours develop. Hueper (31) has denied that this is the invariable sequence of events, but the patho- genesis of bladder tumours under these circumstances has been firmly established by Weil et al (32). The parallel with subcutaneous sarcomas is clear. Because they are injected repeatedly at a high concentration (24}/0) at frequent intervals (once or twice weekly) and always into the same site, some colourings produce local lesions. Once such lesions are formed by long-continued repeated insults to the tissues, development of sarcoma is an almost inevitable outcome. Yet any change in the conditions of administration - the same dose at lower concentrations, less frequent intervals, or injection at a number of sites in rotation - which serves to eliminate or greatly modify the local tissue reaction has a corresponding effect on sarcoma production (22). With increasing understanding of the pathogenesis of this type of tumour there comes a realization that by choosing appropriate conditions of administration one can cause any substance to produce sarcomas or, conversely, not to produce sarcomas. From this assertion two conclusions follow. First, that the precise yield of subcutaneous sarcomas in any rat experiment has no relevance as an indication of carcinogenic potency. In the rat, the incidence of spontaneous tumours of this sort, or of tumours induced by the injection of saline, is so low that it is unjustifiable to regard the production of 18% sarcomas by Indigotine as non-significant while attributing carcinogenicity to Brilliant Blue FCF because the yield of sarcomas was 89}/0 [results quoted from Hansen et al (33)]. The other conclusion which is inescapable