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    RED 2G

    Explanation

         This compound was evaluated by J.E.C.F.A. in 1977.

    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 (Priestly & O'Reilly, 1966). Biliary
    excretion of i.v. administered Red 2G was also reported by Ryan &
    Wright (1961).

         When rabbits were fed 0.5 g per kg/bw of the colour 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%. The ratio of o-aminophenol to p-aminophenol was
    the same for rabbits fed Red 2G and rabbits fed aniline previously
    examined by Parker (1960) indicating that hydroxylation does not
    necessarily precede fission of the azolinkage.

         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).

         Methaemoglobin and Heinz bodies have been observed in acute human
    aniline poisoning by a number of investigators (Rodeck & Westhaus,
    1952; Hughes & Treon, 1954; Friefeldt et al., 1937; Jasinski, 1948).
    Methaemoglobin formation occurred first following by Heinz body
    appearance, the latter being an irreversible process. Heinz bodies are
    formed mostly in vivo but can be studied also in vitro.

         It has been shown that phenylhydroxylamine is the N-hydroxylated
    metabolite which produces most of the methaemoglobin observed in the
    dog after injection of aniline. The reaction probably proceeds by
    oxidation of phenylhydroxylamine to nitrosobenzene by O2 and Hb with
    conversion of Hb to methaemoglobin (Kiese, 1959).

         Since the rat is less susceptible to Heinz body producing agents
    than the cat and possibly man a procedure was devised which increased
    the sensitivity of the rat to these agents by pretreating rats with
    p-aminopropiophenone, 15 mg/kg bw subcutaneously (Unilever, 1974).
    Methaemoglobinaemia is a reversible response to toxic injury and
    depends on the integrity of the erythrocyte in vivo. Reduction of
    methaemoglobin occurs through an NADH-dependent diaphorase system to
    which is deficient in subjects with hereditary methaemoglobinaemia or
    in young infants.

         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-acetamide-1-naphto-
    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 &
    Campbell, 1966; Jenkins et al., 1966).

         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, presumed to be a Sulfur-
    containing compound. Two groups of 12 rats were fed purified diet and
    a purified diet containing 0.51% Red 2G respectively. Faeces were
    collected and it was calculated that 48.2% of the sulfur derived from
    Red 2G was excreted in the faeces (Unilever, 1974).

         The effects of feeding aniline, para-aminophenol and
    phenylhydroxylamine at a level of 0.1% in the diet of rats were
    compared for 13 days. The results indicated that compared with a
    control group, para-aminophenol had no effect on spleen weight,
    aniline increased mean relative spleen weight by about 60% and
    phenylhydroxylamine increased mean relative spleen weight by about
    500%. Examination of the blood after feeding phenylhydroxylamine for
    11 days revealed a high incidence of Heinz bodies. Therefore, the
    toxic effects of feeding aniline can be attributed to
    phenylhydroxylamine which is a metabolite of aniline (Jenkins et al.,
    1966b; Gellatly & Burrough, 1966).

         Three groups of six four-week old female rats were fed in their
    diet 0%, 0.5% Red 2G or 0.093% aniline. All rats were fed ad libitum
    for 19 days. The faeces of rats fed Red 2G were almost black; the
    faeces of rats fed aniline were a normal colour. Both Red 2G and
    aniline caused a similar increase in spleen weight, accelerated
    erythropoiesis and haemosiderin content (Jenkins & Campbell, 1966).

         For 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. For rats fed purified diets containing
    0.004%, 0.006% and 0.012% phenylhydroxylamine for two weeks there was
    a linear relationship between intake of phenylhydroxylamine and
    relative spleen weight (Jenkins et al., 1967a; Gellatly & Burrough,
    1968).

         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 mg 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 mg/ml (blood). At
    all levels of Red 2G fed to rats the proportion of Red 2G metabolized
    to phenylhydroxylamine was constant (Unilever, 1974).

         In another experiment 250 mg/kg bw of Red 2G was administered by
    gastric intubation to 5 male and 5 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 and
    acetylphenylhydrazine as a positive control. No Heinz bodies were seen
    with Red 2G (BIBRA, 1965.)

         No binding of Red 2G to serum protein occurs (Jenkins et al.,
    1966a).

    TOXICOLOGICAL STUDIES

    Acute toxicity

                                                                        

                                    LD50
    Animal        Route             (g/kg)              Reference
                                                                        

    Mouse         Oral          7.35                    Unilever, 1974
                  i.p.          4.76 (4.13-5.85)        Unilever, 1974
                  i.p.          3.0-4.0                 BIBRA, 1965

    Rat           i.p.          6.35 (5.62-7.17)        Unilever, 1965
                  Oral          >5.0                    Unilever, 1974

    Guinea-pig    Oral          4.81 (3.16-7.35)        Unilever, 1974
                  i.p.          3.00 (1.83-4.91)        Unilever, 1974

    Rabbit        Oral          >5.0                    Unilever, 1974

    Chicken       Oral          >10.0                   Unilever, 1974
                                                                        

         No deaths occurred in six weaning rats following administration
    of 5 g/kg bw (Unilever, 1974).

         A dose of 5 g/kg bw was administered on each of two successive
    days to a rabbit weighing 3.8 kg and a dose of 25 g/kg bw 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 (Unilever, 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
    subarachnoid haemorrhage (Unilever, 1974).

         No deaths occurred in three-week old and nine-week old chickens
    following administration of 10 g/kg bw. There was no evidence of renal
    necrosis (Unilever, 1974).

    Short-term studies

    Mouse

         Five 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. Five mice of each sex
    at each dose level were killed at 26, 55 and 96 days, when a full
    autopsy and haematological investigation was carried out on each
    animal. No adverse effect on growth or food consumption was evident in
    any animal given Red 2G in the diet. Heinz bodies were seen at all
    levels, the incidence being related to dose and duration of treatment.
    The maximal effect was seen on day 26 and and day 25 at a level of
    0.1% and below and on day 26 at 1.0 and 2. 2.0%. Splenomegaly was seen
    at 2.0% in both sexes and at 1.0% in females. Increased relative liver
    weights were found in females at 2.0% throughout the tests and at 1.0%
    after 26 days treatment. The only pathological finding attributable to
    the administration of Red 2G was increased haemosiderin in the Kupffer
    cells of the liver at 2.0 and 1.0%. Haemosiderin was also present in
    the spleen at 2.0% throughout the test; at 1.0, 0.1 and 0.01% the
    incidence of haemosiderin increased with the duration of treatment.
    (BIBRA, 1965).

         In another study 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
    macrophages. No toxic effects were observed in mice fed the diet
    containing 0.02% (Unilever, 1974).

    Rat

         Six groups of 5 male and 5 female rats were given diets
    containing 0.0, 0.05, 0.1, 0.5, 1.0 and 2.0% Red 2G for three weeks. A
    further four groups of 10 male and 10 female rats were given diets
    containing 0.0, 0.01, 0.05 and 0.1% Red 2G for two months. Retarded
    growth associated with reduced food consumption was seen at 5.0 and
    2.5% after nine days, with an initial retardation at 2.0%. No effect
    on growth or food consumption was seen at lower levels of
    administration. Macrocytosis, reticulocytosis and polychromasia were
    evident at 5.0% with circulating normoblasts and a normoblastic
    marrow. Heinz bodies were present in animals at 1.0% and above after
    nine days and 1.0% and 0.5% after three weeks' exposure. Signs
    indicative of increased erythropoiesis could be seen in animals at all
    levels down to 0.1%. Significant splenomegaly was evident at all
    levels above 0.5% with scattered non-significant increases in spleen
    weight at 1.0 and 0.05% after two months. Increased kidney weight was
    also seen at 0.1% and above. On histological examination the only
    change attributable to Red 2G was the increased haemosiderin seen in
    the Kupffer cells of the liver, renal tubule cells and spleen in all
    animals at 2.0% and in some at 1.0 and 0.5%. Blood samples were taken
    from the tail of the rats. By nine days a definite haemolytic anaemia
    with Heinz body formation was present at all levels from 0.5 to 2.0%
    (BIBRA, 1965).

         Three groups of 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 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., 1966c; Gellatly et al., 1966).

         Four groups of 24 male and 24 female rats were fed at dietary
    levels of Red 2G which would ensure intakes of 100  and 600  the
    assumed average daily dietary intake of Red 2G. This was achieved by
    feeding sausage meat, containing 0, 30 and 180 parts per million Red
    2G respectively in the diet at a level of 80%. 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
    sausage meat containing 180 parts per million Red 2G, there was
    increased erythropoiesis, increased splenic red pulp haemosiderin and

    increased red pulp reticular impregnation with iron. No effects on
    spleen were seen in rats fed sausage meat containing 30 parts per
    million of Red 2G. Red 2G at 30 parts per million and at 180 parts per
    million in sausage meat had no detectable histological effect on liver
    (Jenkins et al., 1966d).

    Short-term studies on aniline

    Rat

         Aniline dissolved in isotonic saline was administered either
    intravenously to rats under other anaesthesia or by stomach tube.
    Saline was administered to control animals. Blood samples from the
    tail were taken at 30-minute intervals at first and then at 60-minute
    intervals. The no-effect dose of aniline was 20 mg/kg bw orally and
    10 mg/kg bw intravenously (Jenkins & Robinson, 1967).

         Groups of 6 male and 6 female rats were fed diets containing
    0.098% aniline and molecular equivalent levels of p-aminophenol and
    phenylhydroxylamine. Methaemoglobinaemia, Heinz bodies and splenic
    enlargement were noted in rats which received either aniline or
    phenylhydroxylamine. The no-effect single oral dose of anilines in
    rats was 20 mg/kg bw (Jenkins et al., 1970).

    Man

         Aniline was administered orally to a volunteer for five days. The
    dose was 10 mg on days 1 and 2 and 25 mg on days 3, 4 and 5. Urine
    samples were tested for urobilinogen, glucose and protein and blood
    tests included, haemoglobin, methaemoglobin, packed cell volume, serum
    transaminases, alkaline phosphatase, thymol turbidity, serum proteins,
    serum bilirubin and the staining for Heinz bodies. None of these tests
    revealed an effect due to the ingestion of aniline, nor could Red 2G
    be detected in the urine (Jenkins et al., 1967b).

         Single oral doses of 5 and 15 mg aniline had no effect in 20
    human subjects; doses ranging from 25 to 65 mg significantly increased
    the blood level of methaemoglobin to 2.46% but no Heinz bodies were
    observed. Although human subjects are more sensitive to aniline
    in vivo than rats, the methaemoglobin content of rat blood exposed
    to phenylhydroxylamine in vitro exceeded that of human blood exposed
    to phenylhydroxylamine in vitro. Glucose promoted the production of
    methaemoglobin (Jenkins et al., 1970).

    Long-term studies

    Mouse

         Five groups of 40 male and 40 female mice were fed diets
    containing 0, 0.005%, 0.025%, 0.125% and 0.625% for 20 months. Splenic
    enlargement and darkening were seen in mice fed dietary levels of
    0.125% and 0.625% of Red 2G; in these animals there was accelerated
    formation of red blood cells in the spleen and increased deposition of
    iron in spleen and kidneys. There was no evidence of carcinogenic
    attributable to the feeding of Red 2G to mice. More than three-
    quarters of the animals in each group survived for two years
    (Unilever, 1974).

    Rat

         Five groups of 40 male and 40 female rats were fed at dietary
    levels of 0.004%, 0.016%, 0.064% and 0.16% Red 2G. Rats fed diets
    containing 0.064% and 0.16% of Red 2G showed splenic enlargement and
    darkening attributable to increase storage of iron resulting from
    haemolysis of red blood cells. Necrosis of splenic elastica was also
    identified, this lesion being a sequel to prolonged splenic
    enlargement. There was no evidence of carcinogenicity attributable to
    the feeding of dietary levels of Red 2G up to 0.16% for two years.
    Over half of each group of rats survived for two years (Unilever,
    1974).

         Two groups of 30 male and 30 female rats were fed a diet
    containing 0, or 0.5% Red 2G. At the 0.5% level there was enlargement
    and darkening of the spleen, attributable to accelerated splenic
    erythropoiesis, increased splenic haemosiderin deposition and
    extensive degeneration of splenic elastica. Biochemical studies of
    blood and urine revealed no adverse effects on liver and kidneys which
    could be attributed to feeding 0.5% Red 2G in the diet. More than half
    the animals in the test group survived for two years.

    Reproduction studies

         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 onto the same diet. No adverse effects were seen on
    litter size, litter weight and weaning weight nor were there any
    abnormalities at autopsy (Unilever, 1974).

    Comments

         This compound was evaluated in 1977. A temporary ADI for man was
    established at 0-0.006 mg/kg bw. Further work required was a
    multigeneration reproduction/teratology study as well as studies on
    bone marrow to elucidate the toxic effects on erythropoiesis.

         None of the studies requested have been completed. The previously
    established temporary ADI was extended.

         A monograph was prepared.

    EVALUATION

    Estimate of temporary acceptable daily intake for man

    0-0.006 mg/kg bw.

    FURTHER WORK OR INFORMATION

         Required by 1981.

         A multigeneration reproduction/teratology study as well as
    studies on bone marrow.

    REFERENCES

    BIBRA (1965) Research Report No. 3

    Daniel, J. W. (1962) Toxicol. Appl. Pharmacol., 1, 572

    Friefeldt, F., Schilowa, A. & Ludwiriowsky, R. (1937) Folia haemel,
         56, 333

    Gellatly, J. B. M. (1968) Unpublished report to Unilever

    Gellatly, J. B. M. & Burrough, R. (1966) Unpublished report to
         Unilever

    Gellatly, J. B. M. & Burrough, R. (1967) Unpublished report to
         Unilever

    Gellatly, J. B. M. & Burrough, R. (1968) Unpublished report to
         Unilever

    Gellatly, J. B. M., Salmond, G. & Burrough, R. (1966) Unpublished
         report to Unilever

    Hughes, J. P. & Treon, J. F. (1954) Arch. Hyg. and Occup. Med., 10, 192

    Jasinski, B. (1948) Schwerz - Med. Wchschr., 78, 1282

    Jenkins, F. P. & Campbell, P. J. (1966) Unpublished report to Unilever

    Jenkins, F. P. & Robinson, J. A. (1967) Unpublished report to Unilever

    Jenkins, F. P. et al. (1966a) Unpublished report to Unilever

    Jenkins, F. P. et al. (1966b) Unpublished report to Unilever

    Jenkins, F. P. et al. (1966c) Unpublished report to Unilever

    Jenkins, F. P., Salmond, G. & Gellatly, J. B. M. (1966d) Unpublished
         report to Unilever

    Jenkins, F. P. et al. (1967a) Unpublished report to Unilever

    Jenkins, F. P., Salmond, G. W. A. & Robinson, J. A. (1967b)
         Unpublished report to Unilever

    Jenkins, F. P. et al. (1970) Unpublished report to Unilever

    Kiese, M. (1959) Arch. exp. Path. Pharmak., 235, 360

    Parke, D. V. (1960) Biochem. J., 77, 494

    Priestly, B. G. & O'Reilly, W. J. (1966) J. Pharm. Pharmacol., 18, 41

    Rodeck, H. & Westhaus, H. (1952) Arch. Kinderh., 145, 77

    Rofe, P. (1957) Brit. J. industr. Med., 14, 275

    Ryan, A. J. & Wright, S. B. (1961) J. Pharm. Pharmacol., 13, 492

    Unilever (1974) Unpublished review of the biological effects of food
         colour Red 2G dated November 1974

    Walker, R. (1971) Private communication
    


    See Also:
       Toxicological Abbreviations
       Red 2G (WHO Food Additives Series 12)
       Red 2G (WHO Food Additives Series 16)
       RED 2G (JECFA Evaluation)