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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION



    TOXICOLOGICAL EVALUATION OF SOME
    FOOD COLOURS, EMULSIFIERS, STABILIZERS,
    ANTI-CAKING AGENTS AND CERTAIN
    OTHER SUBSTANCES



    FAO Nutrition Meetings Report Series 
    No. 46A WHO/FOOD ADD/70.36




    The content of this document is the result of the deliberations of the
    Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
    27 May - 4 June 19691





    Food and Agriculture Organization of the United Nations

    World Health Organization



                   
    1 Thirteenth report of the Joint FAO/WHO Expert Committee on Food
    Additives, FAO Nutrition Meetings Report Series, in press;
    Wld Hlth Org. techn.  Rep. Ser., in press.


    CHLOROPHYLL COPPER COMPLEX AND CHLOROPHYLLIN COPPER COMPLEX,
    SODIUM AND POTASSIUM SALTS

    Biological Data

    Biochemical aspects

    Oil soluble chlorophyll copper complex has part of the magnesium
    replaced by copper. Water soluble chlorophyllin copper complex has
    part of the magnesium replaced by copper and its methyl and phytyl
    ester groups replaced by sodium and potassium. Any toxic effects are
    therefore, in part, due to free ionisable copper present in the
    complex. Potassium sodium chlorophyllin copper complex (4 per cent.
    total Cu, 0.25 per cent. ionic Cu), in concentrations above 0.1 per
    cent. of the diet, appears as chlorophyllin and Cu ions in the plasm.
    Phylloerythrine is probably not formed in the rat; ingested
    chlorophyll was excreted in the faeces as calcium complex. No copper
    storage occurred in liver, kidney or spleen of rats at dietary levels
    of 0.1 per cent, or 1 per cent. of sodium and potassium chlorophyllin
    copper complex. There was no effect on iron storage at these levels.
    Guinea-pigs fed 0.5 per cent. or rats fed 3 per cent. of the complex
    in their diet showed no evidence of scurvy (Harrison et al., 1954).

    Acute toxicity
                                                                                                     
    Compound                Animal      Route             LD50           Reference
                                                    mg/kg body-weight
                                                                                                 
    Potassium sodium
      chlorophyllin         mouse       oral              7000           Harrison et al., 1954
      copper complex
    Chlorophyllin                       i.p.               190           Harrison et al., 1954
      copper complex
    Sodium chlorophyllin
      copper complex        mouse       i.v.             > 400           Worden et al., 1955
                                        i.m.             > 500           Worden et al., 1955
                                        i.p.            > 1000           Worden et al., 1955
                            rat         i.v.             > 250           Worden et al., 1955
                                        i.m.             > 250           Worden et al., 1955
                                        i.p.             >1000           Worden et al., 1955
                            rabbit      i.v.             > 200           Worden et al., 1955
                                        i.m.              > 60           Worden et al., 1955
                                        i.p.             > 500           Worden et al., 1955
                            cat         i.p.               >60           Worden et al., 1955
                            dog         i.v.             > 200           Worden et al., 1955
                                        i.m.              > 50           Worden et al., 1955
                                        i.p.             > 200           Worden et al., 1955
                            pig         i.v.              > 10           Worden et al., 1955
                                        i.m.              > 20           Worden et al., 1955
                                        i.p.              > 50           Worden et al., 1955
                                                                                                 
    
    Six mice were given 2500 mg/kg body-weight sodium chlorophyllin copper
    complex orally for 7 days without any ill effects (Worden et al.,
    1955). Five male and 4 female rats were fed a diet containing 15 per
    cent. Na K C.C. for 10 days without any adverse effects except weight
    loss related to food refusal (Harrison et al., 1954). Two guinea-pigs,
    2 rabbits, 2 cats and 1 dog were given sodium chlorophyllin copper
    complex 1000 mg/kg body-weight, orally daily for 7 days without any
    adverse effects (Worden et al., 1955).

    Short-term studies

    Mouse. Sodium chlorophyllin copper complex in 0.05, 0.1, 1.0 and 5
    per cent. aqueous solutions was injected daily for 10 days
    subcutaneously at points along the spine followed by exposure of the
    mice to sunlight, UV lamp, or darkness. Many mice died and liver and
    kidney changes, more extensive in irradiated animals, were seen
    (Tomino, 1958).

    Rat. Thirty rats received oral doses of 2000 mg/kg body-weight
    sodium chlorophyllin copper complex for 18 weeks without any adverse
    effects (Worden et al., 1955). Offsprings of 6 female rats fed 1 per
    cent. of potassium disodium chlorophyllin copper complex for 19 weeks
    exhibited locomotory difficulties and skeletal muscle defects (Reber &
    Willigan, 1954).

    Guinea-pig.  Five female guinea-pigs received 0.5 per cent.
    potassium sodium chlorophyllin copper complex in their drinking water
    for 11 weeks without ill effects or pathological change. There was no
    evidence of scurvy (Harrison et al., 1954).

    Fowl. Sixty-day-old chickens received orally 70 mg/kg body-weight
    sodium chlorophyllin copper complex for 6 weeks and 8-year-old fowls
    received 500 mg/kg body-weight for 3 weeks without gross adverse
    effects. The yolk of all eggs laid was coloured an intense green
    (Worden of al., 1955).

    Long-term studies

    Rat. Groups of 40 rats were fed diets containing 0, 0.1, 1.0 and 3
    per cent. of potassium sodium chlorophyllin copper complex (4 -5 per
    cent. total Cu, 0.25 per cent. ionic Cu) over their life span. Growth
    rate, feed efficiency, haematology and urinalysis were comparable to
    the controls. Reproduction showed no impairment of conception. No
    gross or histopathological changes attributable to the potassium
    sodium chlorophyllin copper complex were seen. There was no evidence
    of Cu toxicity or deposition in liver, kidney or spleen (Harrison et
    al., 1954).

    Comments

    The copper in these complexes is firmly bound. Though increased plasma
    levels of copper have been reported there is no significant tissue
    storage nor is there any evidence of destruction of ascorbic acid.
    Chlorophyll copper complex has higher toxicity when given parenterally
    but this has no toxicological significance if this colour is used by
    the oral route, No significant chronic effects were seen in the
    long-term tests in rats. Changes in the copper levels in the plasma
    may be of importance for people suffering from Wilson's disease. At
    present the limit for free copper in the sodium and potassium salts of
    chlorophyllen copper complex is much higher than that in the complex.

    EVALUATION

    Level causing no toxicological effect in the rat

    3 per cent. (= 30 000 ppm) in the diet equivalent to 1500 mg/kg
    body-weight per day.

    Estimate of acceptable daily intake of chlorophyll copper complex for
    man

                                   mg/kg body-weight
    Unconditional acceptance                         
                                       0 - 15

    Estimate of acceptable daily intake of chlorophyllin copper complex,
    sodium and potassium salts, for man

                                   mg/kg body-weight
    Temporary acceptance                             
                                       0 - 15

    Further work required by June 1972

    See tentative specifications, Ref. 19, Annex I.

    REFERENCES

    Harrison, J. W. E., Leoni, S. E. & Trabin, B. (1954) J. Arner. 
    Pharm. Assoc., Sc. Ed., 43, 722

    Reber, E. F. & Willigan, D. A. (1954) Amer. J. vet. Res, 15, 643

    Tomino, U. (1958) Kobe Ika Daigaku Kiyo, 14, 98

    Worden, A. N., Bunyan, J. & Kleissner, M. (1955) Brit. Vet. J.,
    111, 385
    


    See Also:
       Toxicological Abbreviations