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 RED 2G EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA BIOCHEMICAL ASPECTS Rats were injected intravenously with Red 2G. Bile was collected for six hours and analysed. The recovery of the colour was on average 64% of the administered quantity (Priestley and O'Reilly, 1966). Biliary excretion of i.v. administered Red 2G was also reported (Ryan and Wright, 1961), Red 2G (purity 72%) was orally administered in aqueous solution to rats and rabbits, the urine was collected free from faeces over periods of 24 hours and the daily excretion of Red 2G and its metabolites measured. No intact dye could be found in the urine or any hydroxylated derivatives thereof. P-aminophenol was found without preliminary acid hydrolysis. P-aminophenol was also found as the only component after acid hydrolysis of the glucuronic acid fraction. The total amount of p-aminophenol was estimated after hydrolysis with N and 5N HCl. The former hydrolysis both N-acetyl- p-aminophenol and p-amino-phenylsulfate; the latter, in addition, hydrolysis p-aminophenol glucuronide. Forty-eight hours after oral administration of 216 mg of the dye to rats 49% of the dye excreted was, after hydrolysis, found as p-aminophenol; this was 45 and 55% after oral feeding of 720 mg of the dye to rabbits. 5-5.6% and 2% of the dye was excreted as o-aminophenol and aniline, respectively 24 hours after administration of 1 g Red 2G to rabbits. After administration of 1 g Red 20 to rats 0.26% of the dye was excreted as o-aminophenol (Daniel, 1958). When rabbits were fed 0.5 g per kg bodyweight of Red 2G the following metabolites could be identified in urine over a period of 48 hours: total p-aminophenol 46%, p-aminophenylglucuronide 37% and aniline 0.6% and o-aminophenol 9% (Daniel, 1962). The ratio of o-aminophenol to p-aminophenol was the same for rabbits fed Red 2G and rabbits fed aniline previously examined (Parke, 1960) indicating that hydroxylation does not necessarily precede fission of the azolinkage. In another experiment 250 mg/kg body weight of Red 2G was administered by gastric intubation to five male and five female rats. On average the males excreted 61.8% of the dose in the urine and the females 71.5%. 42.2% of the dose was excreted in the urine as p-aminophenol in 48 hours, 9.2% as aniline in 24 hours and 3% as unreduced dye in 24 hours. The corresponding faecal excretion was 6.3%, 1.0% and 0.1%. For females urinary excretion amounted to 56.4% as p-aminophenol in 48 hours, 2% as aniline in 24 hours and 2.6% as unreduced dye in 24 hours. The corresponding faecal excretion was 8.6%, 0.3% and 1.6% (Walker, 1971). In vitro studies were conducted on samples of mouse, rat and human blood with 100 and 200 mg% concentration of Red 2G or acetylphenyl-hydrazine as a positive control. No Heinz bodies were seen with Red 2G (Rofe, 1955). No binding of Red 2G to serum protein occurs (Jenkins et al., 1966a). The contents of a rat caecum were incubated at 37°C with a solution of Red 2G in isotonic saline. At one hour intervals a sample of the incubate was filtered and Red 2G was estimated in the filtrate by measuring the optical density. Two metabolites of Red 2G were detected in the incubation mixture after separation by thin-layer chromatography on silica plates. One was 2-amino-8-acetamido-1-naphthol-3,6-disulfonic acid, the other, aniline, was detected using two different solvent systems. When Red 2G was incubated at 37°C with liver homogenate the same two metabolites were detected (Jenkins et al., 1966b). When a mixture of Red 2G and caecal contents were incubated at 37°C darkening at the surface was observed. This was attributed to oxidation of a metabolite of Red 2G (Jenkins et al., 1966b). Two groups of 12 rats were fed purified diet and a purified diet containing 0.51% Red 2G respectively. Faeces were collected for four days from day 16 and it was calculated that 48.2% of the sulfur derived from Red 2G was excreted in the faeces (Jenkins et al., 1966b). TOXICOLOGICAL STUDIES Special studies on reproduction Two groups of 46 male and 46 female rats were fed 0.2% Red 2G in their diet for 18 weeks and then mated for 10 days. The progeny were weaned on the same diet and mated at 16 weeks and the F2 generation was also weaned on the same diet. No adverse effects were seen on litter size, litter weight and weaning weight nor were there any abnormalities at autopsy (Robinson et al., 1971). Special studies on the metabolites In rats fed purified diets containing 0.1%, 0.2% and 0.3% Red 2G for two weeks there was a linear relationship between intake of Red 2G and relative spleen weight. In rats fed purified diets containing 0.004%, 0.008% and 0.012% phenylhydroxylamine for two weeks there was also a linear relationship between intake of phenylhydroxylamine and relative spleen weight. For individual samples of rat blood and human blood the amount of oxidation of haemoglobin to methaemoglobin was linearly related to the logarithm of phenylhydroxylamine concentration. From the dose-response curve it has been estimated that for rat blood the no-effect dose is between 0.5 and 1 µg phenylhydroxylamine/ml blood. The response of human blood to phenylhydroxylamine was more variable than the response of rat blood. The no-effect concentration of phenylhydroxylamine in vitro for human blood ranged from 0.46 to 4.1 µg/ml. At all levels of Red 2G fed to rats the proportion of Red 2G metabolized to phenyl-hydroxylamine was constant (Jenkins et al., 1966e, 1967; Gellatly and Burrough, 1966, 1967). Acute toxicity LD50 Animal Route (g/kg bw) Reference Mouse Oral 7.35 Anonymous, 1974 i.p. 4.76 (4.13-5.85) Anonymous, 1974 i.p. 3.0-4.0 Anonymous, 1965 Rat i.p. 6.35 (5.62-7.17) Anonymous, 1974 Oral >5.0 Anonymous, 1974 Guinea-pig Oral 4.81 (3.16-7.35) Anonymous, 1974 i.p. 3.00 (1.83-4.91) Anonymous, 1974 Rabbit Oral >5.0 Anonymous, 1974 Chicken Oral >10.0 Anonymous, 1974 A dose of 5 g/kg body weight was administered on each of two successive days to a rabbit weighing 3.8 kg and a dose of 25 g/kg body weight was administered on each of two successive days to a rabbit weighing 4.3 kg. No signs of toxicity were observed and their red cells contained no Heinz bodies (Anonymous, 1974). Histological studies in rats, rabbits and guinea-pigs dosed as given above showed extensive renal necrosis. In mice dosed orally with Red 2G there was gross leptomeningeal vascular engorgement and focal sub-arachnoid haemorrhage (Anonymous, 1974). Short-term studies Mouse Red 2G was fed for six weeks to five groups of 10 mice at dietary levels of 0, 0.02, 0.1, 0.5 and 1.0%, The toxic effects observed were development of Heinz bodies, methaemoglobinaemia, splenic enlargement, accelerated splenic erythropoiesis and increased levels of haemosiderin in splenic macrophages. No toxic effects were observed in mice fed the diet containing 0.02% (Gellatly et al., 1968). Groups of 15 male and 15 female mice were given diets containing 0.0, 0.01, 0.1, 1.0 and 2.0% Red 2G for three months. Five mice of each sex at each dose level were killed at 26, 55 and 96 days. No adverse effects on growth or food consumption were found. At 0.1, 1.0 and 2.0% a dose-related decrease in Hb, RBC and PCV was seen, with some compensation for red cell loss at three months. A dose-related increase in reticulocytes, Heinz bodies, anisocytosis, polychromasia, poikilocytosis and marrow reaction was seen. Considerable amounts of colouring were found in the upper gastrointestinal tract at both 1.0 and 2.0% in both sexes and at the 0.1% level in males: the bile duct contained a dark brown fluid. Enlargement and congestion of the spleen was observed in both sexes at the 2.0% level and at the 1.0% level in females throughout the test. A temporary effect on the relative spleen weight was seen at 0.1 and 1.0% in both sexes at 26 and 55 days. The relative liver weights were constantly significantly increased in females at 2% and scattered, significant increases in relative liver weights affected both males and females at 0.1 and 1.0%. Significant increases in kidney weight were also noted, being temporary at days 26 and 55 at the doses of 0.01, 0.1 and 1.0% but in females present at 2.0% at 96 days. Hyperplasia and excessive haemosiderin were found in the spleen of both sexes, together with an increase in round cell infiltration and pigment in Kupffer's cells in the liver (Anonymous, 1965). Rat Groups of five rats (Carworth Farm E) were given diets containing 0.0, 1.0, 2.5 and 5.0% Red 2G for 90 days. Retarded growth associated with reduced food consumption was seen at 2.5 and 5.0%. A reduction of both Hb and PCV and macrocytosis, reticulocytosis and polychromasia were evident at 5.0% with circulating normoblasts and a normoblastic marrow. At 1% the red cells were of mixed size with increased polychromasia. Heinz bodies were present at all concentrations. Further there was a significant increase in relative weight of spleen in all dome groups and of kidney at 2.5 and 5% (Anonymous, 1965). Groups of five male and five female rats were given diets containing 0.0, 0.05, 0.1, 0.5, 1.0 and 2,0% Red 2G for three weeks. Signs of increased erythropoiesis and enlargement and darkening of the spleen could be seen in animals at all levels down to 0.1%, and even at 0.05% some of the adverse effects could be noticed. The spleen was significantly enlarged at all dietary levels except 0.05% and a significant increase in relative weight of kidneys was seen at 0.5, 1.0 and 2.0%. Increased haemosiderin was found in the liver, spleen and kidneys (Anonymous, 1965). Groups of 10 male and 10 female rats were given diets containing 0.0, 0.01, 0.05 and 1% Red 2G for two months. Signs of erythropoiesis were seen at 0.1% and splenomegaly with congestion in some animals at 0.05 and 0.1% (Anonymous, 1965). Rats were fed 1-1.5 g/kg/day Red 2G for 75 days. The mean peak Heinz body level was 80% falling to a maintained level of 30%. Internal changes included a moderate though well controlled anaemia, pronounced reticulocytosis, and splenomegaly (Rofe, 1957). Three groups o£ 12 rats received Red 2G in the drinking-water at levels of 0%, 0.1% and 0.5% for 100 days. Heinz bodies were seen after 10 days in the red cells of rats fed 0.5% Red 2G in the drinking-water, fewer were seen after 18 days and none or very few on later occasions. A few Heinz bodies were occasionally seen in the red cells of some of the rats fed 0.1% Red 2G in the drinking-water. The spleens of rats fed 0.1% Red 2G were slightly larger than controls and the spleens of rats fed 0.5% Red 2G were very much larger than controls, Histological examination of livers of rats fed 0.5% Red 2G revealed an increase in haemosiderin present in Kupffer's cells and increased erythropoietic activity. Histological examination of the spleens of rats fed 0.5% Red 2G also revealed increased erythropoiesis and red pulp engorgement. There was no effect of Red 2G on urine specific gravity (Jenkins et al., 1966d; Gellatly et al., 1966). Four groups of 24 male and 24 female rats were fed a diet containing purified diet, cooked sausage without colour, cooked sausage with 30 and 80 ppm Red 2G. The cooked sausage was mixed so that the levels of Red 2G were equivalent to 0, 24 and 114 ppm. The feeding was continued for 13 weeks. Food intake and body weight gain were recorded weekly. After nine weeks a liver function test (bromosulfthalein retention test) was carried out. After 12 weeks the refractive index of the urine was measured. Just before the end of the experiment a detailed haematological investigation was done. In the beginning of the experiment after 10-11 days the presence of Heinz bodies was controlled. At post-mortem the weights of the spleen, kidneys, liver, heart and testes were determined. The diets containing Red 2G in sausage meat had no effect on growth, organ function or organ weights. Blood tests also revealed no evidence of toxicity. Histological examination revealed that, in the spleens of rats fed 144 ppm Red 2G with sausage meat, there was increased erythropoiesis, increased splenic red pulp haemosiderin and increased splenic red pulp reticular impregnation with iron. No effects on spleen were seen in rats fed 24 ppm Red 2G with sausage meat. Red 2G at 24 ppm and at 144 ppm in sausage meat had no detectable histological effect on liver. There was some evidence that the diet provided in this experiment was not totally adequate. There was thyroid hyperplasia and loss of hair in many rats. In this experiment 144 ppm of Red 2G had still an effect (Jenkins, 1966c; Gellatly and Burrough, 1968). Long-term studies Mouse Groups of 40 male and 40 female mice (Colworth C57B1) were fed diets containing 0, 0.005, 0.025, 0.125 and 0.625% Red 2G for 80 weeks. Growth, food intake and survival were recorded. Blood tests were performed after three, seven and 80 weeks for the presence of Heinz bodies and at the end of the test also for values of Hb, Ret, WBC, differential cell counts and methaemoglobin. Serum and urine biochemistry were investigated (for urine refractive index Ph, glucose, protein and urea nitrogen). For liver function SGOT, SGPT and SLDH were estimated. The absolute organ weight and the histology of the brain, heart, liver, spleen, kidneys and testes were determined. Heinz bodies, increased methaemoglobin, splenic darkening and enlargement, increased splenic haemosiderin content and accelerated splenic erythropoiesis were seen in mice fed dietary levels of 0.125 and 0.625% of Red 2G. There was no evidence of carcinogenicity attributable to the feeding of Red 2G to mice. More than three-quarters of the animals in each group survived for two years (Jenkins et al., 1971a; Gellatly et al., 1970a, b; Gellatly and Marlow, 1970a). Rat Groups of 40 male and 40 female rats (Colworth wistar) were fed diets containing 0, 0.004, 0.016, 0.064 and 0.16% Red 2G for two years. Growth, food intake and survival were recorded. Blood tests were performed after 11 and 40 days for packed cell volumes and the presence of Heinz bodies and after two years' feeding also for values of Hb, Ret, WBC and differential cell counts. Serum biochemistry (Na, K, Cl, urea nitrogen, creatinine, SGPT, SGOT, SLDH and alkaline phosphatase) was investigated. The absolute organ weight and the histology of the brain, heart, liver, kidneys, spleen, pituitary and adrenals was determined. Splenic darkening and enlargement, with degeneration of splenic elastica, were seen in rats fed dietary levels of 0.064 and 0.16% Red 2G. More than half the animals survived for two years (Jenkins et al., 1971a, b; Gellatly et al., 1970b, c; Gellatly and Marlow, 1970b; Billing and Jenkins, 1968; Robinson, 1968; Groger and Robinson, 1969). REFERENCES Anonymous (1965) Acute and short-term feeding studies on Red 2G with associated haematological investigations, British Industrial Biological Research Association, Research Report 3/1965. Unpublished report submitted to WHO Anonymous (1974) Acute toxicity by oral intubation, intraperitoneal injection of Red 2G in several animal species. Summary of toxicological data prepared by J. M. Philp, Research Division, Unilever Ltd, submitted to WHO by Unilever Ltd Billing, C. C. and Jenkins, F. P. (1968) Chronic toxicity of Red 2G; 2-year rat feeding trial (0 and 0.5% Red 2G), biochemical studies after feeding for 180 days. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Daniel, J. W. (1958) The metabolism of azo dyes. V. Azo-geramine (benzeneazo-8-acetamide-l-naphthol 3; 6-disulphonic), October 1958, Report No. IHR/126. Unpublished report from Industrial Hygiene Research Laboratories, submitted to WHO by Imperial Chemical Industries (ICI) Daniel. J. W. (1962) The excretion and metabolism of edible colours, Toxic Appl. Pharmac., 4, 572 Gellatly, J. B. M., Salmond, G. and Burrough, R. (1966) Sub-acute toxicity of Red 2G; pathology. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Gellatly, J. B. M., Jenkins, F. P., Salmond, G., Robinson, J. and Marlow, K. (1968) Sub-acute toxicity of Red 2G in mice. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Gellatly, J. B. M., Conniff, R. A. and Lovell, M. J. (1970a) Chronic toxicity of Red 2G; 2-year rat feeding trial (0-0.16% Red 2G), macroscopic pathology of rats killed at termination of the trial, Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Gellatly, J. B. M., Lovell, M. J. and Conniff, R. A. (1970b) Chronic toxicity of Red 2G: 80 week mouse feeding trial, macroscopic pathology of mice killed at termination of the trial. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Gellatly, J. B. M., Marlow, K. and Salmond, G. (1970c) Chronic toxicity of Red 2G; 80 week mouse feeding trial, pathology of mice dying or killed during the trial. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Gellatly, J. B. M., Marlow, K. and Salmond, G. (1970d) Chronic toxicity of Red 2G; 2-year rat feeding trial (0-0.16% Red 2G), pathology of rats dying or killed during the trial. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Gellatly, J. B. M. and Burrough, R. (1966) Effect of diets containing aniline and metabolites of aniline on spleen weights; pathology. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Gellatly, J. B. M. and Burrough, R. (1967) Pathology of Red 2G and phenylhydroxylamine in rat diets. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Gellatly, J. B. M. and Burrough, R. (1968) Toxicity of Red 2G; effect of feeding to rats in a sausage meat diet for 90 days; pathology. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Gellatly, J. B. M. and Marlow, K. (1970a) Chronic toxicity of Red 2G; 80 week mouse feeding trial, histopathology of mice killed at termination of the trial. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Gellatly, J. B. M. and Marlow, K. (1970b) Chronic toxicity of Red 2G; 2-year rat feeding trial (0-0.16% Red 2G), histopathology of rats killed at termination of the trial. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Jenkins, F. P., Campbell, P. J., Robinson, J. and Salmond, G. (1966a) Metabolism of Red 20 (protein binding capacity). Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Jenkins, F. P., Salmond, G., Campbell, P. J,, Hardy, J. and Robinson, J. (1966b) Metabolism of Red 20 by rat liver homogenate. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Jenkins, F. P., Salmond, G. and Gellatly, J. B. M. (1966c) Sub-acute toxicity of Red 20 in sausage meat. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Jenkins, F. P., Salmond, G., Robinson, J., Campbell, P. J. and Gellatly, J. B. M. (1966d) Sub-acute toxicity of Red 20. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Jenkins, F. P., Robinson, J., Gellatly, J. B. M., Salmond, G. and Campbell, P. J. (1966e) Effect of diets containing aniline and metabolites of aniline on spleen weights. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Jenkins, F. P., Robinson, J., Gellatly, J. B. M. and Salmond, G. (1967) Effect of Red 20 and phenylhydroxylamine on spleen weight. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Jenkins, F. P., Robinson, J., Salmond, G. and Groger, W. (1971a) Chronic toxicity of Red 2G; 80 week mouse feeding trial, living animal studies. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Jenkins, F. P., Robinson, J., Salmond, G. and Groger, W. (1971b) Chronic toxicity of Red 20; 2-year rat feeding trial (0-0.16% Red 20), living animal studies. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Parke, D. V. (1960) Biochem. J., 77, 494-503 Priestley, G. and O'Reilly, W. J. (1966) Protein binding and the excretion of some azo dyes in rat bile, J. Pharm. Pharmacol., 18, 41-45 Robinson, J. (1968) Chronic toxicity of Red 20; 2-year rat feeding trial (0 and 0.5% Red 2G), biochemical studies after feeding for 200 days. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Robinson, J. and Paragreen, G. (1971) Reproduction in rats fed Red 2G. Unpublished report from Unilever Research Laboratories, submitted to WHO by Unilever Ltd Rofe, P. (1955) Induction of Heinz bodies by edible azo dyes in the rat. Unpublished report from Industrial Hygiene Research Laboratories, submitted to WHO by Imperial Chemical Industries (ICI) Rofe, P. (1957) Azo dyes and Heinz bodies, Brit. J. industr. Med., 14, 275-280 Ryan, A. J. and Wright, S. E. (1961) The excretion of some azo dyes in rat bile, J. Pharm. Lond., 13, 492 Walker, R. (1971) Private communication to E. M. den Tonkelaar, National Institute of Public Health, Bilthoven, Netherlands
See Also: Toxicological Abbreviations Red 2G (WHO Food Additives Series 14) Red 2G (WHO Food Additives Series 16) RED 2G (JECFA Evaluation)