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    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 37C 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 37C 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 37C
    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 g
    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 g/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)