INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION SUMMARY OF TOXICOLOGICAL DATA OF CERTAIN FOOD ADDITIVES WHO FOOD ADDITIVES SERIES NO. 12 The data contained in this document were examined by the Joint FAO/WHO Expert Committee on Food Additives* Geneva, 18-27 April 1977 Food and Agriculture Organization of the United Nations World Health Organization * Twenty-first Report of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 1977, WHO Technical Report Series No. 617 BROWN FK EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA BIOCHEMICAL ASPECTS After an intraperitoneal dose to a rat of 1.5 g/kg body weight the extremities become orange in 60 minutes, and the animal sluggish. The urine was normal. After 24 hours the animal was normal but urine was deep orange-yellow (Goldblatt and Frodsham, 1952). After an intraperitoneal dose to a rat of 1.5 g/kg bw the extremeties become orange in 60 minutes, and the animal sluggish. The urine was normal but urine was deep orange-yellow (Goldblatt and Frodsham, 1952). On incubation with the contents of rat ileum and caecum Brown FK and its components undergo azo-reductive fission with formation of sulfanilic acid, a phenazine-like material (P) and ill-defined products that can be separated chrometographically. Brown FK, in common with other brown azo coloutings, also undergoes azo-reductive fission when incubated with rat-liver homogenate, but P has not been detected among the products. Oral administration of Brown FK to rats, pigs, rabbits and guinea-pigs results in the excretion of sulfanilic acid in urine and faeces; P is detectable in trace/small amounts in faeces, but is mainly present in caecal contents, predominantly during the first six hours after dosing. A "blue material" is excreted in the urine. On intraperitoneal administration to rats, Brown FK initially gives rise to brown colouring in bile; later, sulfanilic acid and the "blue material" appear in the urine. P is not found in faeces or in caecal contents. As formed in vitro from Brown FK, P is a complex mixture comprising two main components P1 and P2. P1 has been identified as 1,4,7-tri-aminophenazine and P2 is probably a methylhomologue of P1 (Fore and Walker, 1967; Fore, Walker and Goldberg, 1967). The metabolism of Brown FK would be extremely difficult to study since the final metabolite mixture from the many components would be exceedingly complex. Investigations have therefore been carried out on the major components: 1,3-diamino-4-(p-sulfophenylazo)benzene ("azo-benzene" component) and 2,4-diamino-5-(p-sulfophenylazo)toluene ("azo-toluene" component). The metabolism of both components was qualitatively similar, a proportion being excreted unchanged but the bulk reductively cleaved to sulfanilic acid and the corresponding amine (the latter being acetylated before excretion). Although the metabolism of only these two components of Brown FK has been studied,there is no reason to suppose that the metabolism of the other Brown FK components should be fundamentally different. The primary metabolic reaction in each case would be expected to be cleavage of the azo linkages. A summary of the products of azo reduction of the six major components of Brown FK is shown below (Howes, 1969; Munday and Kirkby, 1969).
Metabolism of the "azotoluene" component of Brown FK in the rat Preliminary examnation of urine from rats fed the "azotoluene" component of Brown FK showed the presence of sulfanilic acid and small quantities of unchanged dye. Examination of an extract of this urine revealed the presence of 5-acetamido-2,4-diaminoteluene (the major metabolite), 2,5-diacetamido-4-aminotoluene, 2,4-diacetamido-5-amino- toluene and 4,5-diacetamido-2-aminotoluene. Unchanged dye was again identified in faecal extracts; no other dye-derived compounds were detected. The metabolism of the "azotoluene" component of Brown FK is summarized below: (Munday 1969).
An attempt was made to determine whether the "azobenzene" component of Brown FK is reductively cleaved in humans, as in rats. Reduction of the closely-related compound, protosil rubrum (XIII) has been shown to take place in human subjects (Fuller, 1937).
Administration of the "azobenzene" component of Brown FK to human subjects led to no detectable unchanged dye in the urine, and no appreciable urinary sulfanilic acid. It can be inferred from these results that the "azobenzene" component is not absorbed from the intestine as such, but they give no information on the possible reduction of this compound in vivo, since it was shown that orally administered sulfanilic acid was not absorbed in man. Sulfanilic acid, if formed from the dye, would therefore be excreted in the faeces; the experimental confirmation of this would be technically very difficult and these studies were not pursued further (Jenkins and Favell, 1971). 1,2,4-triaminobenzene and 2,4,5-triaminotoluene have been shown to uncouple oxidative phosphorylation in vitro, interfering with ATP production in the muscle cell and to ionic imbalance with cell death (Munday, 1971). Acute toxicity Animal Route LD50 mg/kg body weight Reference Mouse oral >2 000 (with salt) Grasso et al., 1968 oral 1 100-2 250 (with salt) Edwards and Wilson, 1966 oral 960-1 140 (no salt) Edwards and Wilson, 1966 i.p. 1 500-2 000 (with salt) Grasso et al., 1968 i.p. 960-1 720 (with salt) Edwards and Wilson, 1966 i.p. 840- 880 (no salt) Edwards and Wilson, 1966 Rat oral > 8 000 (with salt) Grasso et al., 1968 oral 900-1 910 (with salt) Edwards and Wilson, 1966 oral 780- 970 (no salt) Edwards and Wilson, 1966 i.p. 750-1 150 (with salt) Grasso et al., 1968 i.p. 1 100-2 250 (with salt) Edwards and Wilson, 1966 i.p. 960-1 150 (no salt) Edwards and Wilson, 1966 Guinea-pig oral 3 000 (with salt) Edwards and Wilson, 1966 oral 2 610 (no salt) Edwards and Wilson, 1966 i.p. 900 (with salt) Edwards and wilson, 1966 i.p. 780 (no salt) Edwards and Wilson, 1966 Rabbit oral 450- 680 (with salt) Edwards and Wilson, 1966 oral 390- 590 (no salt) Edwards and Wilson, 1966 Chicken oral > 10, 000 (with salt) Edwards and Wilson, 1966 oral >8 700 (no salt) Edwards and Wilson, 1966 For all species, animals dying did so from within a few minutes to 96 hours. Many animals, after either oral or intraperitoneal treatment, showed lack of coordination, hypersensitivity and hyperactivity; convulsions usually preceded death (Edwards and Wilson, 1966). Meningeal congestion or haemorrhage was seen at post-mortem examination in rats and mice which died following both oral and intraperitoneal treatment with 3.4 g/kg of Brown FK as a 10% solution. This was the highest dose administered and the condition may have been present to a lesser degree in animals treated with lower levels of Brown FK but postmortem identification of the lesion was made difficult by tissue coloration. The meningeal congestion/haemorrhage was probably caused by the sodium chloride in the dye solution since we have observed this lesion after administration of hypertonic solutions of sodium chloride to rats. In this instance, the lowest dose levels at which the meningeal lesion has been observed were 6.0 g/kg of sodium chloride orally as a 10% solution and 4.0 g/kg intraperitoneally as a 5% solution (Edwards and Wilson, 1966). Following oral intubation, external tissue coloration was apparent after some hours in rats and guinea-pigs. No coloration of the tissues was seen in rabbits and chickens. After intraperitoneal injection, external tissue coloration was apparent and intense after a few minutes in the rats and guinea-pigs. Colour was seen in the faeces of rats, mice, rabbits and guinea- pigs up to 24 hours a£ter oral treatment; it was also excreted in the urine of rats, mice, guinea-pigs and rabbits within 15 minutes of either oral or intraperitoneal treatment (Edwards and Wilson, 1966). Hearts from some rats and mice surviving for 21 days after treatmant were examined histologically. A degenerative lesion was found in 15% of rats given orally 1-2.5 g/kg body weight but not with 3.37 g/kg. The same lesions were found in mice in 50% given orally 0.9 g/kg but not if given 0.6, 1.35 and 2.03 g/kg. If given intraperitoneal 25-60% of mice showed lesions at 0.75 and 1.03 g/kg body weight (Edwards and Wilson, 1966). Amines derived from Brown FK and from its two myotoxie components, 2,4-diamino-5-(p-sulfophenylazo)toluene and 1,3-diamino-4- (p-sulfo-phenylazo)benzene were injected intravenously into rats in single doses of 3.13-25 mg/kg. The mixture of amines from Brown FK was also injected into mice in the same range of doses. Cardiac and muscular lesions were produced by the amines in both species. These amines are biological degradation products in the intestine. The finding that orally administered Brown FK is myotoxic in rats but not in mice is probably due to differences in the intestinal flora in the two species (Walker, Grasso and Gaunt, 1970). Investigation of the pigment deposited After feeding Brown FK to rats and mice pigment is found in heart, skeletal muscle, tongue, diaphragm, thyroids, brain, liver, kidneys, spleen, lungs, pancreas, bladder, testes, ovary, uterus, skin, stomach, duodenum, ileum, brown fat and bone marrow. In addition, a pigment has been detected in the plasma of rats. Staining tests commonly used to identify lipofuscin were negative with the exception of the test for metachromasia with toluidine blue. The tests applied were as follows: Usual response of Response of Brown FK known lipofuscin induced pigment Test for iron negative negative Sudan fat stains positive negative Reduction of ferric salts positive negative Reduction of ammoniacal silver salts positive negative Basophilic properties positive negative Periodic acid - schiff reaction positive negative Acid fastness acid fast negative Toluidine blue at pH 3 stains greenish metachromatically green Two further histochemical tests clearly differentiate between lipo-fuscin and the Brown FK - induced pigment: - Potassium permanganate/oxalic acid bleached lipofuscin, but not the brown FK - induced pigment. - Sodium dithionite bleached both lipofusein and the Brown FK induced pigment. However, after rinsing and allowing to stand in air, the Brown FK - induced pigment reappeared; lipofusein was permanently bleached. The Brown FK - induced pigment does not fluoresce in ultra-violet light. All the samples of lipofuscin which we have examined were fluorescent. Pigment has been found in the thyroid, brown fat and bone marrow; these tissues have not been recorded as being sites for lipofuscin deposition. Furthermore, a coloured substance has been demonstrated in the plasma, never lipofuscin. The speed at which the Brown FK - induced pigment is deposited is uncharacteristic of lipofuscin information. In acute studies, pigment has been seen in the intestinal wall and villi within 24 hours of feeding the dye, and in the kidney after five days. Pigment masses produced in macrophages either in vivo after the intraperitoneal injection of Brown FK into mice, or in vitro, when Brown FK was incorporated in the macro-phage culture medium, appeared identical. Tests for lipofuscin proved negative; the pigment in the macrophages closely resembled that seen in macrophages in stained sections of tissues taken from rats and mice fed Brown FK. Electron microscope studies have identified differences in morphology between lipofuscin and the Brown FK - induced pigment. In aged rats and mice fed Brown FK, conjugate forms were observed in which induced pigment and control lysosomal material appeared in the same membrane limited body (Hope, 1971). It is likely that this compound oxidises within the cell to 1,4,7-triaminophenazine
This would also explain the behaviour of the pigment with sodium dithio-nite. The latter reduces the phenazine ring to 5,10-dihydroderivative which is probably colourless. After exposure to air reoxidation would occur. Thus the pigment may not represent evidence of sub-lethal cell damage, but is more an insoluble oxidation product of a dye metabolite. 1,2,4-triaminobenzene was very rapidly oxidized to 1,4,7-triamino-phenazine by a mitochondrial suspension; no phenazine derivatives were detected with triaminotoluene under the same circumstances (Kirby, 1968). 1,4,7-triaminophenazine is a brown, water-insoluble material, which is very readily formed from 1,2,4-triaminobenzene (Muller, 1889). Short-term studies Mouse Groups of 10 male and 10 female mice received the colour (both fresh and stored) at the level of 1 g/kg daily for three weeks. A significant reduction in weight gain was noted in the mice receiving the stored solution but not in those receiving fresh solution. One male and one female receiving the fresh solution showed cardiac lesions (BIBRA, 1964). Daily oral or intraperitoneal doses up to 2 g/kg or 1 g/kg respectively for 43 days to groups of 10 or 12 mice were well tolerated (Grasso et al., 1968). Groups of 10 male and 10 female mice (Colworth C57 B1 strain, initially six weeks old) were fed for 90 days on a synthetic diet containing 0, 0.05, 0.075, 0.10, 0.25, 0.50, 0.75, 1.0 and 2.0% of Brown FK (equivalent to 0, 0.025, 0.0375, 0.05, 0.125, 0.25, 0.375, 0.50 and 1% Brown FK coloured components) composition 51% colour, 47% salt. A further group of 20 mice were fed synthetic diet containing added 1.0% sodium chloride as a control for the additional dietary salt derived from the Brown FK. At the 0.125% dietary colour level pigment deposition occurred in tissues. At 0.25% and above there was splenic enlargement, at 0.5% liver and heart were also enlarged and at 1% there was reduced growth, poor food utilization, liver, spleen, heart and testicular enlargement and histological evidence of degenerative heart lesions. Thyroids, muscle, intestine and squamous part of stomach were pigmented (Ashmole et al., 1958). Rat Groups of animals received the colour at the level of 0.5 g Brown FK per kg body weight for three weeks, orally or intraperitoneally. When dosed orally, 20 rats were treated and six animals died between five and 11 doses. Post-mortem examination of rats dying during the test or killed at the end revealed general tissue staining in five rats. Of 18 hearts examined histologically, eight showed degenerative lesions and a brown pigment was observed in small amounts in nine hearts after three weeks. In the multiple dose intraperitoneal test, eight rats were treated and none died during the treatment period. General organ staining was observed in all animals at post-mortem examination. Hearts from seven rats were examined microscopically and degenerative lesions were found in one heart and small amounts of pigment in three hearts after three weeks (Kirkby, 1968). No ill-effects were seen in three weanling rats given a 0.1% solution for 28 days, the intake being 15 mg/day (Goldblatt and Frodsham, 1952). Administration of two or three oral doses of 1 g/Brown FK/kg bw to rats induced a myopathy in cardiac and skeletal muscles characterized by multiple vacuoles about 1-2 µ in diameters. Ultrastructurally, these were shown to consist of areas of fibrillolysis. Histochemically, the myopathy was accompanied by a moderate increase in acid-phosphatase activity and by a loss of phosphorylase activity. Subsequently complete lysis of the affected fibres ensued. In the heart, lysis was followed by macrophage invasion and fibroblastic proliferation, and in skeletal muscle by regeneration. The occurrence of lipofuscin in muscle fibres and in macrophages was scanty and erratic. When Brown FK was given in the diet at a level of 2%, fibrillolysis and an increase in the number and electron-density of lysosomes was observed ultrastructurally during week two to three of the test. These changes were accompanied by a marked elevation of histochemically demonstrable acid phosphatase. Progressive deposition of lipofuscin was the principal pathological feature during week three to 12 (Grasso et al, 1968). Daily oral doses up to 2 g/kg for 43 days to groups of 10 rats induced rapid loss of weight and death, with severe damage to cardiac and skeletal muscle, characterized by vacuolar myopathy and lipofuscin deposition. Of three pure components of Brown FK studied, one (2,4-diamino-5-(p-sulfophenylazo)toluene) and to a lesser extent another (1,3-diamino-4-(p-sulfophenylazo)benzene) produced similar, but not identical lesions to those induced by the parent colouring after repeated oral doses of 0.5 g/kg. Ultrastructural studies confirmed the extensive loss of myofibrillar elements and histochemical studies revealed a loss in the activity of mitochondrial enzymes. Similar intraperitoneal injections in doses up to 1.0 g/kg for 43 days to groups of 10 or 12 rats did not have any effect on the heart or skeletal muscle (Grasso et al., 1968). In a series of experiments using groups of 10-12 rats receiving the colour at levels of 1 and 0.1 g/kg orally and 1, 0.25 and 0.1 g/kg intraperitoneally daily up to a maximum of 43 doses the following observations were made. A specific cardiac lesion was identified at the oral dose of 1 g/kg. There were large areas of myocardial necrosis and replacement by large mononuclears, with involvement of the sub-pericardial region and endocardium. Some myocardial cells had lost their stainable cytoplasm and appeared only as empty sheaths. Intraperitoneally, the colour produced little or no cardiac damage at any dose tested. At these high doses of 1 g/kg, most animals showed congestion, fatty change or necrosis of the liver with hydropic degeneration of the kidney. There was no obvious splenomegaly. Daily doses of 100 mg/kg by stomach tube produced two pericardial and one sub-pericardial lesions. In addition, early hydropic degeneration of the kidney was seen in two rats with one of those animals also showing fatty change in the liver (BIBRA, 1964). Administration of Brown FK (purity 80.0%) at dietary levels of 0, 0.001, 0.01, 0.1 and 1.0% for 150 days showed no adverse effects on growth, food consumption, haematological indices, liver and kidney function and organ weights. One male rat at the 1.0% level showed the typical myocardial changes, other rats showed deposits of lipofuscin especially in females. The no-effect level was 0.1% (Gaunt et al., 1968). Groups of 12 male and 12 female rats (Colworth Wistar strain, initially three to four weeks old) were fed for 112 days on a commercial stock diet, containing 0, 0.05, 0.1, 0.5, 1.0 and 2.0% of Brown FK (0, 0.025, 0.05, 0.25, 0.5 and 1% Brown FK coloured components) 51% dye component, 47% salt. A further group of 24 rats were fed the commercial stock containing an added 1% sodium chloride as a control for the additional dietary salt derived from the Brown FK. In addition, to eliminate damage to the heart from cardiac puncture in any rat kept to 16 weeks, groups of six male and six female rats were fed for six weeks on powdered stock diet containing 0, 0.05, 0.5 and 2.0% of Brown FK (0, 0.025, 0.25 and 1.0% Brown FK coloured components). A group of 12 rats also received 1.0% sodium chloride added to the basic diet. All these rats were used for biochemical tests during weeks 0-6 after which they were killed. At the 0.25% level tissue pigmentation appeared, at 0.5% liver enlargement occurred and at the 1% level there was reduced growth, poor food utilization, enlargement of liver, testes and thyroid, histological evidence of degenerative heart lesions, increased urinary indican excretion and elevation of SGOT. The intestine and squamous portion of stomach as well as thyroid were stained. The no effect level was 0.05% (Ashmole et al., 1966). Pig Groups of female and male pigs were given doses of 0, 100, 250 and 500 mg/kg/day for 24 weeks without adverse effects on growth, food consumption, haematological indices, liver and kidney function and organ weights. Lipofuscin was widely distributed in both sexes at all dose levels in one or more organs. It particularly affected the liver, where it was accompanied by increased lysosomal enzyme activity, more marked at the higher dose levels. It was also seen in the heart in males and here it was associated with an increased acid-phosphatase activity, and in the kidneys, at the highest dose level in females and at all levels in males. A no-effect level was not seen (Gaunt et al., 1968). Special studies on "azobenzene" and "azotoluene" components Mouse Groups of three male and three female mice (C57 B1) were fed 0, 0.5 and 1.0% of components in a synthetic type diet for six weeks. With both compounds the thyroids were dark and intestine and squamous portion of stomach were stained salmon pink. Heart lesions were seen in all mice fed 1% azotoluene and not in those given azobenzene. More pigment was seen in mice fed azobenzene component, little in those fed azo-toluene component (Kirkby, 1968). Rat Groups of three male and three female Colworth Wistar rats were fed azobenzene and azotoluene component at dietary levels of 0, 0.5 and 1% in commercial stock diet for six weeks. The thyroids of rats on "azo-benzene" were dark, heart, muscle and brain were stained but the intestine was stained only slightly. Pale hearts and meningeal haemorrhage were seen with "azotoluene", otherwise pigmentation was as with "azobenzene". One-sixth "azobenzene" and 4/5 "azotoluene" rats had heart lesion. Most pigment was seen histologically in "azobenzene" rats, least in "azo-toluene" rats (Kirkby, 1968). In another study 1,2,4-triaminobenzene was given to groups of six to seven rats orally five days per week for two weeks at 50, 60, 75 and 100 mg/kg body weight/day. Six-sevenths receiving 100 mg/kg/day died after three doses with severe heart lesions, 7/11 on 75 mg/kg/day also died after three doses. Heart pigmentation occurred after five days treatment or longer. Animals on lower doses showed both extensive heart pigmentation and cardiac necrosis. 1,2,4,5-tetraaminobenzene was given to groups of six rats orally five days per week for two weeks at 150 and 200 mg/kg body weight/day. 1,2,3,4-tetraaminobenzene was given to groups of six rats orally five days per week for two weeks at 125 and 166 mg/kg body weight/day. No frank heart lesions and only instances of diffuse increase in interstitial cells in the heart were observed. No heart or thyroid pigment deposition was seen (Mulky et al., 1969). Long-term studies Mouse Groups of 40 male and 40 female Colworth C57 B1 mice were fed for 80 weeks on a synthetic diet containing 0, 0.0125%, 0.0375%, 0.075%, 0.125% and 0.625% Brown FK coloured components (Brown FK purchased contained 62.5% coloured components). Only at the 0.625% was there reduced growth and food utilization and higher mortality among females. There was increased liver, kidney, spleen, brain and testes weight, evidence of splenic haemopoiesis, increased myocardial fibrosis. Heart weight was increased at the 0.125% level. Increased hepatic nodules were seen as from 0.075% and pigment deposition as from 0.0375% level. At termination, after 80 weeks, the number of animals with nodules for the different dose levels was 26, 23, 27, 56, 42 and 64 respectively. Increased hepatic nodules were observed at dose level 0.075% and higher. The number of mice with hepatocellular carcinoma were 3, 2, 0, 5, 6 and 2 respectively. Pigment deposition was observed at dose level 0.0375% and higher (Wilson et al., 1970). Rat Groups of 32 male and 36 female Colworth Wistar rats were fed for two years on a synthetic diet containing 0, 0.01%, 0.03%, 0.06%, 0.1% and 0.5% of Brown FK coloured components (Brown FK purchased contained 54.2% coloured components). Only at the 0.5% level was there increased splenic weight and hepatic granulomata. Pigment deposition was seen as from 0.06%. The no-effect level for pigment deposition was 0.03% and 0.06% when based on toxicity evidence (Wilson et al., 1971). Special studies on mutagenicity Brown FK and its constituents were assayed for mutagenicity in Salmonella typhimurium TA 1535, TA 1537 and TA 1538 when activated by a rat liver supernatant fraction. Mutagenicity was linearly dose- dependent in the range 0-3 mg/plate with activities ranging from 22 to 50 times the spontaneous mutation frequency. One sample of Brown FK was mutagenic in the absence of metabolic activation producing a 16-fold increase in mutation at 4 mg/plate. Two major constituents of Brown FK, 2,4-diamino-5-(p-sulfophenylazo)toluene (I) and 1,3-diamino- 4-(p-sulfo-phenylazo)benzene (II) each present at about 18% in the complete colour, were mutagenic in TA 1538. Mutagenicity was linearly dose-related in the range 0-1 µmol/plate, with slopes of 0.35 mutants/nmol for compound I and 1.5 mutants/nmol for compound II. This activity was dependent on metabolic activation. Four other major constituents were inactive, as was sulfanilic acid, the major excretion product. The mutagenicity of Brown FK could be largely accounted for by the combined effects of compound I and II (Venitt and Bushell, 1976). REFERENCES Ashmole, R. T., Campbell, P., Kirkby, W. W. and Wilson, R. (1966) Effects of feeding dietary Brown FK to rats for six and 16 weeks. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Ashmole, R. T., Kirkby, W. W. and Wilson, R. (1958) Thirteen week mouse feeding trial. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Edwards, K, B. and Wilson, R. (1966) Acute toxicity of Brown FK in rats, mice, guinea-pigs, rabbits and chickens. Unpublished report from Unilever Research Laboratories Fore, H. and Walker, R, (1967) Studies on Brown FK. I. Composition and synthesis of components, Fd. and Cosmet. Toxicol., 5, 1-9 Fore, H., Walker, R. and Golberg (1967) Studies on Brown FK. II. Degradative changes undergone in vitro and in vivo, Fd. and Cosmet. Toxicol., 5, 459-473 Fuller, A. T. (1937) Lancet, 194 Gaunt, I. F., Hall, D. E., Grasso, P. and Golberg, L. (1968) Studies on Brown FK. V. Shortterm feeding studies in the rat and pig, Fd. and Cosmet. Toxicol., 6, 301-312 Goldblatt and Frodsham (1952) Private communication from ICI (unpublished report) Grasso, P., Muir, A., Golberg, L. and Batstone, E. (1968) Cytopathic effects of Brown FK on cardiac and skeletal muscle in the rat, Fd. and Cosmet. Toxicol., 6, 13-24 Grasso, P., Gaunt, I. F., Hall, D. E., Golherg, L. and Batstone, E. (1968) Studies on Brown FK. III. Administration of high doses to rats and mice, Fd. and Cosmet. Toxicol., 6, 1-11 Hope, J. (1971) Ultrastructure of the pigment induced in various tissues of the rat by long-term feeding of the dye Brown FK. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Howes, D. (1969) Metabolism of 14C labelled 1,3-diamino-4- (p-sulpho-phenylazo) benzene, a component of the dye Brown FK, in the rat, Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Jenkins, F. P. and Favell, D. J. (1971) Metabolism of the "monoazobenzene" component of Brown FK in human subjects. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Kirkby, W. W. (1968) Effects of Brown FK and two of its constituents on pigment deposition and lesions in rats and mice. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Kirkby, W. W. (1968) Nature of the pigment induced in tissues of rats and mice fed Brown FK. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Mulky, M. J., Munday, R., Ashmole, R. T. and Kirkby, W. W. (1969) Evaluation of the terminal causative agent in Brown FK induced myopathy and pigment deposition. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Muller, E. (1889) Chem. Ber., 22, 856 Munday, R. (1969) Metabolism of 2,4-diamino-5-(p-sulphophenylazo) toluene. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Munday, R. (1971) Uncoupling of oxidative phosphorylation by Brown FK metabolites. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Munday, R. and Kirkby, W. W. (1969) Metabolism of 1,3-diamino-4- (p-sulpho-phenylazo) benzene. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Venitt, S. and Bushell, 0. T. (1976) Mutagenicity of the food colour Brown FK and constituents in Salmonella typhimurium, Mutation Research, 40, 309-316 Walker, R., Grasso, P. and Gaunt, I. F. (1970) Myotoxtcity of amine metabolites from Brown FK, Fd. and Cosmet Toxicol., 8, 539-542 Wilson, R., Gellatly, J. B. M., Kirkby, W. W. and Ashmole, R. T. (1970) Biological evaluation of Brown FK: 80-week mouse feeding trial. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd. Wilson, R., Gellatly, J. B. M., Kirkby, W. W. and Ashmole, R. T. (1971) Biological evaluation of Brown FK: 2-year rat feeding trial. Unpublished report from Unilever Research Laboratories, submitted to the World Health Organization by Unilever Ltd.
See Also: Toxicological Abbreviations Brown FK (WHO Food Additives Series 20) BROWN FK (JECFA Evaluation)