THE TOXICOLOGY OF ARTIFICIAL COLOURING MATERIALS 423 result of the red'uctive fission of azo linkages in water-soluble colourings has been studied by Radomski and Mellinger (4) who showed that amines so formed in the rat were absorbed, metabolized and excreted in the same manner as the identical amino compounds given in free form by stomach tube. The most characteristic compound split off in this way is sulphanilic acid, which is absorbed and emerges in the urine as the free acid and its N-acetyl derivative (5). By administering the colouring by the oral route in some rats and intraperitoneally in others, one can judge the extent to which intestinal degradation takes place. Thus when Tartrazine is given by mouth, sulphanilic acid appears in the urine but no free colour- ing is excreted in urine or faeces. When the compound is given parenterally the animal is dyed bright yellow and free colouring appears in the urine, but without sulphanilic acid (6). Whatever the extent to which a colouring is absorbed from the intestine, a complicating factor is the proportion of biliary excretion, which provides a direct route from the liver back into the intestine. Daniel (7) and Ryan and Wright (8) have shown that some water-soluble azo colourings are excreted almost quantitatively in bile. In a study of the relation of protein binding to biliary excretion, Priestly and O'Reilly (9) concluded that preferential binding to liver proteins, as against plasma proteins, was the determining factor - at least in the case of the colours studied by them. Biliary excretion has the effect of recycling the intact colouring, or the products derived from it, through the intestine, a process termed "enterohepatic circulation." The stability of halogenated derivatives of fluorescein, given by mouth to rats, has been studied by Webb et al (10). Of the di-, tri- and tetra- halogenated colours, only the 4-iodo and 4-bromo derivatives were dehalo- genated to fluorescein. Recoveries of unchanged material in the faeces were almost quantitative with tetrahalogenated derivatives, but fluorescein and its dihalogenated derivatives were cleared more slowly than the others. Webb and Brouwer (11) found that increasing halogenation of fluorescein diverts excretion from the urine to bile and hence the faeces, but also increases the total excretion. Elevation of serum protein-bound iodine in man has been quoted as evidence that erythrosine is deiodinated after ingestion (12). How- ever, since no effort was made to distinguish between protein-bound erythrosine-iodine and protein-bound iodine, this conclusion is unconvinc- ing. Sulphonated colourings that are not subject to attack by the

424 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS intestinal micro-organisms are sufficiently strong acids not to undergo appreciable absorption from the intestine. Thus the triarylmethane colourings are excreted almost quantitatively in the faeces (13). Never- theless, some absorption does take place. Hess and Fitzhugh (13) found that less than 5% of the dose of colouring admi'•istered by mouth appeared in rat, rabbit or dog bile, probably reflecting the excretory pathway of absorbed colouring. Although it is stated that none of the colours were found in the urine, one wonders whether re-examination of the question by up-to-date techniques might not reveal the presence of metabolites in urine. In the liver Lipid-soluble azo compounds do not undergo cleavage in the intestine but are absorbed intact and acted upon by liver azo reductase to form the corresponding primary amines (14). Other changes undergone by such azo compounds involve protein-binding, hydroxylation and other effects such as N- and O- dealkylation brought about by microsomal processing (drug metabolizing) enzymes. The subsequent fate of amines formed in the liver, or absorbed from the intestine, depends to a large extent on whether they are sulphonated and thus water-soluble, or whether they are alkyl-substituted anilines subject to extensive transformation within the body (15,16). In the former case they tend to be excreted intact in the urine or via the bile or they may undergo minor conjugation such as acetylation before urinary excretion. The lipophilic amines are subjected to hydroxylation, or oxidation of alkyl substituents which probably proceeds through hydroxy- lation. In the blood such acetylated amines or hydroxyamines exercise their well-known effect in inducing methaemoglobinaemia. This outcome ß has been recorded in man on numerous occasions following exposure to aniline or to "aniline dyes," as in children consuming coloured crayons. Methaemoglobinaemia is only the initial and, within limits, readily re- versible change. Further transformation of haemoglobin leads to the development of erythrocytic inclusions termed Heinz bodies, with increased destruction of the affected red cells, the development of anaemia, reper- cussions on the bone-marrow and deposition of haemosiderin in the spleen and liver. It is certainly true that in many instances these effects are only observed with high doses of colouring. Before one seeks to reach a