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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION



    TOXICOLOGICAL EVALUATION OF SOME
    FOOD COLOURS, ENZYMES, FLAVOUR
    ENHANCERS, THICKENING AGENTS, AND
    CERTAIN FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES 6







    The evaluations contained in this publication were prepared by the
    Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
    4-13 June 19741


    World Health Organization     Geneva     1975






              

    1  Eighteenth Report of the Joint FAO/WHO Expert Committee on
    Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 557.
    FAO Nutrition Meetings Report Series, 1974, No. 54.

    CHLOROPHYLLIN COPPER COMPLEX, POTASSIUM AND SODIUM SALTS

    Explanation

         These compounds have been evaluated for acceptable daily intake
    by the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
    Ref. No. 20) in 1969.

         Since the previous evaluation additional data have become
    available and are summarized and discussed in the following monograph.
    The previously published monograph has been expanded and is reproduced
    in its entirety below.

    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% total Cu,
    0.25% ionic Cu), in concentrations above 0.1% of the diet, appears as
    chlorophyllin and Cu ions in the plasma. 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% or 1% of sodium and potassium
    chlorophyllin copper complex. There was no effect on iron storage at
    these levels. Guinea-pig fed 0.5% or rats fed 3% of the complex in
    their diet showed no evidence of scurvy (Harrison et al., 1954).

    TOXICOLOGICAL STUDIES

    Acute toxicity

         Six mice were given 2500 mg/kg bw sodium chlorophyllin copper
    complex orally for seven days without any ill effects (Worden et al.,
    1955). Five male and four female rats were fed a diet containing 15%
    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, two
    rabbits, two cats and one dog were given sodium chlorophyllin copper
    complex 1 000 mg/kg bw orally daily for seven days without any adverse
    effects (Worden et al., 1955).

    Acute toxicity
                                                                        

                                       LD50
    Compound          Animal   Route   (mg/kg bw)  Reference
                                                                        

    Potassium sodium  Mouse    Oral      7 000     Harrison et al., 1954
    chlorophyllin
    copper complex

    Chlorophyllin              i.p.        190     Harrison et al., 1954
    copper complex

    Sodium            Mouse    i.v.      > 400     Worden et al., 1955
    chlorophyllin
    copper                     i.m.      > 500     Worden et al., 1955
    complex
                               i.p.    > 1 000     Worden et al., 1955

                      Rat      i.v.      > 250     Worden et al., 1955

                               i.m.      > 250     Worden et al., 1955

                               i.p.    > 1 000     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
                                                                        

    Short-term studies

    Mouse

         Sodium chlorophyllin copper complex in 0.05, 0.1, 1.0 and 5% 
    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 bw sodium
    chlorophyllin copper complex for 18 weeks without any adverse effects
    (Worden et al., 1955). Offspring of six female rats fed 1% 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% 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 bw sodium
    chlorophyllin copper complex for six weeks and eight-year-old fowls
    received 500 mg/kg bw for three weeks without gross adverse effects.
    The yolk of all eggs laid was coloured an intense green (Worden et
    al., 1955).

    Long-term studies

    Rat

         Groups of 40 rats were fed diets containing 0, 0.1, 1.0 and 3% of
    potassium sodium chlorophyllin copper complex (4-5% total Cu, 0.25%
    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.

    EVALUATION

    Level causing no toxicological effect

         Rat: 3% (= 30 000 ppm) in the diet equivalent to 1500 mg/kg bw.

    Estimate of acceptable daily intake for man

         0-15 mg/kg bw*,**

    FURTHER WORK OR INFORMATION

         Required by June 1978

         Revision of tentative specification.

    REFERENCES

    Harrison, J. W. E., Iconi, 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

              

    *    Temporary.

    **   As Na + K chlorophyllin copper complex.


    See Also:
       Toxicological Abbreviations