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

THE TOXICOLOGY OF ARTIFICIAL COLOURING MATERIALS 429 is that there is no basis for the argument that, in choosing safe colourings, less hazard is likely to be associated with the use of colourings that do not produce sarcomas than with the use of those that do. Superficially reason- able though it may be, this attitude simply evades the issue and perpetuates current misconceptions. Undoubtedly some true carcinogens do give rise to local tumours on subcutaneous injection but in such cases their carcino- genicity has invariably been established by other means. Effects on reproduction, toxicity to the foetus and teratogenicity Very little information is available in this area of toxicology, except for acid disazo dyes such as Try_pan blue, Evans blue and various types of Niagara blue. An excellent review has been published (34). While this group of dyes finds no application in foodstuffs, and probably none in cosmetics, the possibility exists that action on foetal development is an attribute of some part of the molecule of the disazo dyes. Beaudoin and Pickering (35) suggested that 1-napththylamine sulphonated in two positions, preferably 3 and 6, is a basis for teratogenicity. Accordingly Christie (36) tested the effect of 1,7diamino-8-naphthol-3,6-disulphonic acid given by the subcutaneous route to rats on day 8 of pregnancy. The maternal deaths, resorptions and developmental retardations observed were attributed to renal toxicity of the compound in the mothers rather than to any direct effect on the embryo. The present position, therefore, appears to be that foetal toxicity and teratogenic activity are confined to a special class of dyes given by injection in a variety of species. The relevance of these results to cosmetics colourings is doubtful. Mutagenesis The observation by German workers that Erythrosine had a slight but nevertheless genuine mutagenic effect on E. coli was followed up by Lfick et al (37) who tested a variety of xanthene and other colourings. Xanthene itself, eosine, eosine BNX and erythrosine showed a very slight but statistically significant mutagenic effect on the bacteria when tested at a concentration of 10-4M, supposedly the same order of magnitude as that used in foodstuffs. Erythrosine at 1%, and Rhodamine B at a 0.5% concentration had distinct mutagenic effects. Induction of mutation in yeast has been reported by Nagai (38), using basic triphenylmethane dyes (such as methyl violet and p-rosaniline) and xanthenes (Pyronines Y and B). Although concentrations as low as 1 ppm or less produced effects, it is questionable whether these results have any significance from the standpoint of mammalian toxicology.