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CLAYSON, Lawson, Santana and Bonser (1965) suggested that in the mouse the oral administration of chemical bladder carcinogens induced hyperplasia of the bladder epithelium in the first days or weeks of the experiment. Subsequently, Clayson and Pringle (1966) showed that the number of mitoses in the normal adult mouse bladder epithelium is very low and suggested that it is necessary to increase the mitotic rate in order to induce tumours. They showed that the implantation of a paraffin wax or cholesterol pellet, or a small glass bead, into the lumen of the bladder increased the mitotic rate. Subsequently, Clayson, Pringle and Bonser (1967) found that a single oral administration of 4-ethylsulphonylnaphthalene-1-sulphonamide, a murine bladder carcinogen, greatly increased the number of mitoses in the bladder epithelium, while Wood (personal communication) observed a smaller increase in mice given 2-acetamidofluorene in the diet. Thus, the correlation of early hyperplasia and subsequent malignancy can be explained on the grounds of an initial increase in the number of mitoses in the bladder epithelium.In the course of the experiments of Clayson et al. (1965) a number of chemicals whose carcinogenicity to the mouse was not known were tested for the ability to induce early hyperplasia of the bladder epithelium. Three of them, di-n-butylnitrosamine, 3-methoxy-2-aminodiphenylene oxide and 2-aminodiphenylene oxide, were effective. It was decided to test the latter for carcinogenic activity in the mouse under a variety of conditions. 2-Aminodiphenylene oxide was shown by Hackmann (1956) to induce a papilloma of the bladder epithelium and 4 malignant tumours of different tissues in 10 rats which survived feeding for from 42 to 89 weeks. Miller, Miller, Sandin and Brown (1949) gave the acetyl derivative -to their Holtzman Albino rats and obtained 5 mammary gland carcinomas and 2 acoustic gland tumours in 7 female, and 1 acoustic gland tumour in 7 male rats when the experiment was terminated after 8 months.4-Aminodiphenyl induced hyperplasia of the bladder epithelium when given by stomach tube but not when dispensed in the diet. It gave only 2 cancers of the bladder in 12 mice surviving to 90 weeks (Clayson et al., 1965). It was decided -to investigate it further. MATERIALS AND METHODSMale and female C57 x IF F1 hybrid mice were bred in the laboratory. All mice were vaccinated against ectromelia. Oxo Diet 41B and water were provided ad libitum. Treatment was started at approximately 12 weeks of age.

JuLL (1951) suggested that the surgical introduction of a pellet containing a test chemical into the lumen of the mouse bladder might be useful for routine testing for carcinogenic activity. The method, it was thought, would possess the following advantages: (i) the chemical would be slowly eluted from the pellet and would therefore remain in contact with the bladder epithelium for a prolonged period; (ii) the metabolic processes of the liver, etc., would be by-passed, and (iii) the bladder would function under approximately normal conditions.Bladder implantation has been used successfully in Leeds Jull, 1958, 1963), in London (Allen, Boyland, Dukes, Horning and Watson, 1957) and in Madison (Bryan, Brown and Price, 1964a, b).The advantages predicted for the technique have not been completely fulfilled. Chemicals have been shown to diffuse from pellets at different rates (Bryan, Brown, Morris and Price, 1964), although there was no correlation between rate of diffusion and carcinogenicity of a series of chemicals. The bladder epithelium has been shown to be permeable to certain chemicals (Bryan, Morris and Brown, 1965;Pringle, 1966), and so it cannot be assumed that metabolism by the liver with consequent excretion of metabolites is necessarily excluded by the use of bladder implantation. Furthermore, the presence of a foreign body, the pellet, in the bladder lumen affects the response of the epithelium to a carcinogen (Bryan and Springberg, 1966) probably because it induces mitosis in the bladder epithelium (Clayson and Pringle, 1966). The pellet by itself usually leads to a background incidence of tumours (Bonser et al., 1958).The purpose of this paper is to present new information on the testing of chemicals by bladder implantation and to reassess the utility of the method.The chemicals investigated consist of aromatic amines and their derivatives, hydrocarbons and dyestuffs. MATERIALS AND METHODSAnimals.-C57 x IF F1 hybrid mice were bred in the laboratory and maintained on Oxo Diet 41B and water, ad libitum. They were 10-12 weeks of age at the start of the experiment.Bladder implantation was carried out by the method of Jull (1951) as modified by Allen et al. (1957). Except where otherwise stated, the experiments were terminated at 40 weeks and the bladders prepared for histology in the usual way. The histological grading of the tumours was assessed by the criteria of Bonser and Jull (1956).