THE TOXICOLOGY OF ARTIFICIAL COLOURING MATERIALS 425 decision on what constitutes a maximum no-effect level, however, it is necessary to consider the following points: (a) The sensitivity of the method used to measure methaemoglobin (bearing in mind the fact that the rat normally has about 1 g methaemo- globin/100 ml blood). (b) The species differences in "resistance" to methaemoglobinaemia, presumably a reflection of the activity of methaemoglobin reductase (17). In this respect the cat is among the most sensitive species, man is almost as sensitive and the rat is a highly resistant species. Human genetically- determined deficiency of red-cell glucose 6-phosphate dehydrogenase creates a particular susceptibility to haemolytic amines, nitro compounds and the like (18). (3) Similar variations in degree of susceptibility to the formation of Heinz bodies. In our studies on food colourings, care has been taken to use a sensitive method for measuring methaemoglobin. In short-term feeding experi- ments carried out on rats, decreasing dietary levels of some colourings have given rise to Heinz bodies after increasingly long latent periods. At still lower dietary levels, although no effect was apparent, the application of a provocative test developed in our laboratories (19) revealed that a latent tendency to Heinz-body formation was still present. This example illustrates the errors that might have arisen from a mechanical, run-of- the-mill approach to safety evaluation, having as its objective the establish- ment of lack of toxicity rather than the discovery of the full facts. A comparison of the rat's susceptibility to the three isomeric dimethyl- anilines and to the trimethylanilines mesidine and pseudocumidine (derived from Ponceau 3R) has revealed the greater potency of the trimethylanilines in producing haemolytic anaemia, methaemoglobinaemia and Heinz bodies (20). General systemic toxicity Gastro-intestinal effects, and specifically catharsis, was an early concern and led to the prohibition of some colours for food use. On a chronic basis in animals, impaired weight gain and liver and kidney damage have often been the most striking effects brought about by high doses. Liver damage may be associated in long-term experiments with the production of liver rumours, which will be referred to below. Observations that present problems in interpretation are, firstly, the finding of an increase in relative liver weight in the absence of histopatho- logical evidence of liver damage, even at the highest dietary level tested

426 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS and, secondly, the presence of pigment granules in reticuloendothelial cells, especially of the liver, and/or in epithelial cells of the proximal convoluted tubules of the kidney. The significance of liver enlargement under these circumstances has been discussed (21). Storage of pigment granules that are neither haemosiderin, lipofuscin nor melanin, but probably represent the colouring in its original or a modified form, may reasonably be inter- preted as an effect of exposure to high doses. In such cases there is no reason to believe that under the conditions of intended use any storage of this sort is likely. Evidence of the uptake of certain dyes (not food colourings) in lysosomes lends particular point to the occasional occurrence of increased amounts of lipofuscin, particularly in the liver cells, after long-term feeding experi- ments. In the past such accumulations of what was called "wear and tear pigment" were ignored. Nevertheless the possibility exists that they represent the remains of previously-existing autophagic vacuoles which had been engaged in the task of restoring the integrity of damaged parenchymal cell cytoplasm. Be that as it may, it must be realized that present-day methods of assessing toxicity to the liver, by means of tests of liver function and observation of histopathological changes, leave something to be desired - especially when the problem is one of detecting low-grade chronic irrever- sible damage. Moreover, the laboratory animal exposed to such agents as colours is not simultaneously exposed to the multitude of environmental toxic hazards by which man is surrounded in everyday life, and which may in certain circumstances exercise a synergistic or even a potentiating effect on hepatotoxic action (22). Tests involving topical application The availability of excellent reviews (23,24), makes it unnecessary to deal here with questions of methodology. Mention should, however, be made of special problems, such as hair dyes, application about the eye, inclusion of colours in dental products, and tests on abraded skin. The effects of colouring matehals on skin and mucous membranes of experimental animals are at most a preliminary guide to further tests of the complete formulation in animals and man. Consequently it is not proposed to deal with the subject further here. CARCINOGENICITY Tumour production by azo compounds has been the subject of many reviews. Clayson (25) distinguishes dimethylaminoazobenzenes. other