IN a careful examination of the long-term effects of large doses of lead, Zollinger (1953) described the tumours of the kidney in rats induced by repeated injection of lead phosphate. This finding has been confirmed by Walpole (personal communication in Matthews and Walpole, 1958) who also obtained tumours in the kidney by injection of lead phosphate. The tumours were similar to those caused by administration of 4-amino-5-fluorobiphenyl (Matthews and Walpole, 1958).Van Esch, van Genderen and Vink (1959, personal communication) fed rats on a diet containing 1 per cent of basic lead acetate over a period of two years. At the end of this period the surviving rats were killed and many were found to have malignant tumours of the kidney. That rats should survive for two years on a diet containing 1 per cent lead acetate is surprising, in view of the known toxicity of lead compounds. In this paper, the findings of van Esch, van Genderen and Vink (1959, personal communication) are confirmed. Fairhall and Miller (1941) had previously carried out experiments with much lower concentrations of lead. They maintained rats on diets containing 0.1 per cent of lead arsenate and 0-1 per cent lead carbonate for two years. The mortality in the group fed the lead carbonate was almost the same as in the controls. At the end of the two year period, the kidneys of the rats were found to have many swollen cells with large vesicular nuclei and brown granules; the latter were most prominent in the proximal convoluted tubules, but no neoplasms were reported. The concentration of lead in the kidney was higher than in the liver but lower than in bone. Lead compounds are known to cause increased excretion of porphyrins by interfering with haemoglobin metabolism. As the porphyrins pass through the kidney they might be the immediate carcinogen, rather than the administered lead. Lead is deposited in bones, so that if the lead itself were carcinogenic then bone tumours would be expected. The hypothesis that porphyrins might be the immediate carcinogens in the kidney was tested by administering allylisopropylacetylcarbamide (Sedormid), which also causes porphyrinuria, to rats. The porphyrin concentration in the urine of rats dosed with lead acetate, sedormid and other substances known to cause porphyrinuria was measured. ExperimentalTwo groups of 20 male 10-week-old Wistar rats were maintained on a 20 per cent protein diet of the following composition; white flour, 68-7 per cent ; casein, 11-5 per cent; milk powder, 8 per cent; margarine, 3*3 per cent; chalk, 1-3 per cent; salt mixture (Glaxo Laboratories), 0 8 per cent; " Bemax ", 2 5 per cent;

IN the course of a series of tests of iroii-containing compounds and irolicomplexes for carcinogenicity, it was found that a preparation of rat-ferritin induced not only a high incidence of sarcomata at the site of repeated subcutaneous injection, but also testicular atrophy, Leydig-cell hyperplasia and benign Leydigcell tumours (Haddow, Dukes and Mitchley, 1961 In this paper the induction of sarcomata at the site of subcutaneous injectiol-i of " rat-ferritin " or cadmium sulphate in rats is described and the failure to induce similar tumours in mice bv injection of the same agents, reported. In an accompanying paper (Roe et al., 1964) the testicular and pituitary changes following the injection of eadmi-Lun sulphate, or ferritin. are described and discussed.MATERIALS AND ',,%IETHODS Rats : Male albino rats of the Chester Beatty straiii were used. In the first experiment courses of injection were begun when the rats were 3-4 weeks old (80-1 00 g.), and in the third experiment when they were 6-7 weeks old (I 70-200 g.).(Rats of this strain are exceptionally large.) The rats were housed in metal cages in groups of 10, fed cubed Diet 86 on 2 days of each week and white bread plus milk, cod liver oil, margarine, or marmite on the 5 other davs. Water was provided ad libitum,

THE discovery of the N-hydroxylation of 2-acetamidofluorene in vivo (Cramer, Miller and Miller, 1959) had been followed by recognition that 4-acetamidobiphenyl (Miller, Wyatt, Miller and Hartman, 1961b) and 2-naphthylamine (Boyland, Manson and Nery, 1960;Troll and Nelson, 1961) are metabolised by similar routes. The carcinogenic aromatic amines are also metabolised to ortho aminophenols, some of which have been shown to be carcinogenic by the technique of bladder pellet implantation in mice (Bonser, Bradshaw, Clayson and Jull, 1956;Allen, Boyland, Dukes, Horning and Watson, 1957), but 2-naphthylhydroxvlamine, the N-hydroxy derivative of 2-naphthylamine, has induced a higher incidence of bladder tumours than any other compound tested (Bonser, Boyland, Busby, Clayson, Grover and Jull, 1963). The N-hydroxy derivatives of 2-acetamidofluorene and 4-acetamidobiphenyl appear to be proximate carcinogens when given intraperitoneally to rats (Miller, Miller and Hartman, 1961a;Miller, Wyatt, Miller and Hartman, 1961b).The present paper describes the potent carcinogenicity of the 2-naphthylhydroxylamine in comparison with that of the parent amine following multiple intraperitoneal injections in rats. A preliminary report of this work has already appeared (Boyland, Dukes and Grover, 1961). (Boyland, Manson and Nery, 1962). The hydroxylamine, which crystallized as cream-coloured plates (m.p. 137°) was stored in the dark at 40 until required for injection. 2-Naphthylamine and an unidentified red-coloured oxidation product were shown to be present as impurities by descending paper chromatography (Whatman No. 1) in light petroleum (b.p. 60-80°); acetone 4: 1. This fast-running solvent system has the advantage that paper chromatograms can be completed without decomposition of the hydroxylamine, and it has been found useful as a solvent for the thin-layer chromatography of naphthylamine derivatives on silica gel (Manson, unpublished work). Attempted purification of 2-naphthylhydroxylamine by recrystallization from chloroform (Baudisch and Fiirst, 1917) yielded a less pure product of lower melting point.Two groups each of 16 male Chester Beatty random inbred strain albino rats (200 g.) were used. Group A received intraperitoneal injections of 2-naphthyl-4

Images Fig. 11 Figs. 12, 14 Fig. 13

IN an accompanying paper the induction of sarcomata at the site of repeated subcutaneous injection into rats of cadmium sulphate, or of cadmium-precipitated rat-ferritin, is described, and the failure to induce such tumours in mice by similar treatment reported (Haddow et al., 1964). In the present paper the occurrence of testicular lesions and of pituitary changes in cadmium-treated rats and mice is described and discussed.For some time it has been known that cadmium is highly toxic to the testes of a number of animals. Parizek and Zahor (1956) reported complete necrosis of the testes of rats given one subcutaneous injection of cadmium chloride in a dose as small as 0.02 millimole per kilogram of body weight. They obtained similar results in mice, rabbits, guinea pigs and golden hamsters. This work has since been confirmed by a number of other workers, including Meek (1959), using mice.Kar and Das (1960) studied the sequence of events after a cadmium chloride injection in rats and noticed intense va'scular congestion of the testis within six hours. After two days the seminiferous epithehum was completely destroyed and transformed into a mass of debris. The changes in the interstitium, which they described as being of " similar magnitude ", resulted within two to seven days in total loss of anatomical structure and functional activity. The present authors obtained similar results in male rats using a single subcutaneous injection of cadmium, sulphate (Cd S04-4H20) in doses varying from 0.5 to 2-0 mg. per 100 g. body weight (equivalent to 0.2 to 0-8 mg. per 100 g. cadmium).A number of reports have commented upon the return of androgenic activity to testes rendered necrotic by cadmium and have described proliferation beneath the tunica albuginea of fibroblast-like cells which later show the typical structure of Leydig ceRs.The

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ALTHOUGH Maisin and Picard (1924) induced cancer of the bladder by implanting pellets containing tar into the bladders of rats, Bonser, Clayson and Jull (1951) developed the technique of bladder implantation in mice, which as modified by Allen, Boyland, Dukes, Horning and Watson (1957) Onie of the difficulties of the method is the choice of a suitable medium with which to mix the substances to be tested. Most apparently inert materials induce tumours in a proportion of mice and even the most active carcinogens do not induce cancer in all the mice treated. For this reason a few substances which would be expected to be biologically inert have been tried. METHODS AND MATERIALSThe technique used was the same as that described by Allen et al. (1957). With practice it is possible to expose the bladder and insert the pellet through a small incision in the skin. The incision may be so small that it can be closed with a single stitch. The probability that the incidence of tumours was due to chance was calculated by the x2 test. Stock mice bred in the Chester Beatty Research Institute were used.Cholesterol (Roche Products), and stearic acid (B.D.H.) were recrystallised from ethanol. Hexadeconal (cetyl alcohol) and octadecanol were purchased from British Drug Houses Ltd. RESULTS AND DISCUSSIONTurmour Induction with " Inert " VehiclesThe incidence of tumours with inert substances some of which might be used in place of paraffin wax or cholesterol are given in Table I. Magnesium stearate gave a low incidence of tumours but it has the disadvantage that in the presence of chelating agents (e.g. ortho aminophenols and 8-hydroxyquinoline) the magnesium forms chelates. If such agents are being tested they would therefore be present as chelates.

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