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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.

    CARAMEL COLOUR (AMMONIA PROCESS)

    Explanation

         These compounds have been evaluated for acceptable daily intake
    by the Joint FAO/WHO Expert Committee on Food Additives (see Annex I,
    Ref. No. 19) in 1971.

         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

         Caramel refers to a large number of poorly defined and complex
    products formed from various carbohydrates generally by heating with
    any of a wide range of acids, bases and salts, under varying
    conditions of temperature and pressure. It might be argued that
    caramel can be considered as a natural constituent of the diet as it
    can be formed when certain foods are cooked or when sucrose is heated.
    Toxicological discrimination is unwarranted between such caramel and
    caramels produced commercially from food-grade carbohydrates, with the
    exception of caramels prepared by processes using ammonia or ammonium
    salts.

         The production of violent hysteria and convulsions in cattle and
    sheep fed ammoniated sugar-containing feed supplements (nitrogen
    content 4-6%), at 6-25% of their rations led to the discovery of the
    presence of about 20% of pyrazines and 10% of imidazoles in these
    ammonium-treated molasses (WHO, 1970). 4-methylimidazole has been
    shown to be the most likely toxic component being a convulsant to
    rabbits, mice and chicks at oral doses of 360 mg/kg bw (Nishie et al.,
    1969). The pyrazines, on the other hand, are mild CNS depressants and
    weak anticonvulsants (Heyns, 1971). Analysis of food grade caramel
    colours, however, showed that only 0.002-0.02% of 4-methylimidazole
    is present in commercial products (Heyns, 1970). Commercial caramel
    colours of undefined origin contain 50-500 ppm 4-methylimidazole
    (Heyns, 1971) while other examinations have shown ranges of
    100-700 ppm (Battelle Memorial Institute, 1971). It has been shown
    that the yields of imidazole compounds increased linearly with the
    increment of molar ratio of ammonia to glucose (Komoto, 1962). Further
    analyses for 4-methylimidazole were being carried out on a large
    variety of caramel colours (Nishie et al., 1971). Generally caramel
    colours contain 50% digestible carbohydrate, 25% non-digestible
    carbohydrate and 25% of melanoidins also found in roasted coffee,
    broiled meats and baked cereal products.

         In groups of two to four rats, the absorption of the colour-
    giving components of caramel was determined by faecal extraction.
    Recoveries varied widely for the 10 or 20% caramel solutions examined
    despite pretreatment for 100 days before testing. About one-third
    of the colour-giving components appeared to be absorbed but no
    conclusions could be drawn regarding the absorption of colourless
    components (Haldi & Wynn, 1951).

    TOXICOLOGICAL STUDIES

    Special studies on reproduction

    Rat

         Fifteen male and 15 female rats were given 0 or 10% caramel
    solution as their sole fluid source until day 100 and were then mated.
    The F1 generation was weaned and given again 0 or 10% caramel
    solution until day 100. It showed no adverse effects as regards
    litter number, haematology, growth, food consumption, gross and
    histopathology (Haldi & Wynn, 1951). A further reproduction study
    including teratology is planned (CIVO, 1972).

    Acute toxicity

                                                                        

                     LD50
    Animal   Route   (mg/kg bw)                       Reference
                                                                        

    Rat      Oral    > 2.3 ml approx. or = 1 900      Foote et al., 1958

             Oral    > 25 ml approx. or =  17 500     Chacharonis, 1960

             Oral    > 30 ml approx. or =  20 400     Chacharonis, 1963
                                                                        

         No abnormalities were detected after observation of animals for
    14 days following administration of 12 different caramel colour
    products mostly based on ammonia or ammonium sulfate catalysts (Foote
    et al., 1956; Chacharonis, 1960; Chacharonis, 1963). A single dose of
    up to 10 g/kg bw in mice and 15 g/kg in rabbits of caramels produced
    by the ammonia catalyser closed pan process or sodium hydroxide
    process did not cause convulsions or other signs of distress
    (Sharratt, 1971).

    Short-term studies

    Rat

         Groups of five male and five female rats were given 1 ml/kg bw of
    concentrated caramel colour for 21 days. Some diarrhoea was induced
    in all animals but no other abnormalities were noted.  Gross and
    histopathology revealed no significant changes due to administration
    of the test compound (Foote et al., 1958).

         Groups of five rats received either 10 or 20% caramel solution
    equivalent to about 10 or 20 g/kg bw as sole source of fluid for 127
    days. Only dark faeces and very mild diarrhoea were noted. No adverse
    effects were noted regarding general health, body weight, food and
    fluid consumption, haematology, gross and histopathology (Haldi &
    Wynn, 1951).

         Six groups of five male and five female weanling rats received 0
    or 10% caramel solution as their sole fluid source for 100, 200 or 300
    days respectively. No adverse effects were noted regarding growth,
    food and fluid intake, haematology, gross and histopathology (Haldi &
    Wynn, 1951).

         Groups of 16 male and 16 female rats received either 0 or 10%
    caramel solution for 100 days and groups of five rats received 20%
    caramel solution for 100 days. At the lower test level there were no
    observable abnormalities as regards growth, food consumption,
    haematology, gross and histopathology. Only growth and haematology
    were examined at the higher test level (Haldi & Wynn, 1951).

         Three groups of 20 male and female rats received either 0 or
    11-14 g/kg bw of caramel solutions for 100 days. Growth and food
    intake did not differ significantly between test and control animals.
    Gross and histopathology showed no abnormal findings related to
    administration of the test compound (Haldi, 1958).

         Four groups of 10 male and 10 female rats received 0, 0.1, 1.0
    and 10% of caramel colour in their diet for 12 weeks. No adverse
    effects were noted on growth, food consumption, urinalysis,
    haematology, gross and histopathology related to administration
    of the caramel colour (Prier, 1960).

         Groups of 10 male and 10 female rats received 0, 5, or 10 g/kg
    caramel colour in their diet for three months. Weight gain was normal
    in all groups. Food consumption, haematology and urinalysis were
    comparable. Gross and histopathology showed no test-related adverse
    findings (Chacharonis, 1960).

         Four groups of 10 male and 10 female rats received 0, 5, 10 and
    20% of two different caramel colours in their diet for 90 days. In 

    addition, a paired feeding study involving five male rats in two
    groups was run for 23 days with one sample at the 20% level, and there
    was no difference in the rate of growth. The only effects attributable
    to treatment were a mild depression in growth of male rats at the 10
    and 20% level due to impalatability of the test diet. No other adverse
    findings were noted in growth, behaviour, mortality, haematology,
    urinalysis, gross pathology, organ weights, and histopathology (Kay &
    Calandra, 1962).

         Four groups of 10 male and 10 female rats received either 0 or
    10% of three different caramel colours in their diet for 90 days.
    Weight gains showed slight reduction compared with controls but food
    consumption was normal for all groups. No abnormalities were noted
    regarding haematology, urinalysis, gross and histopathology
    (Chacharonis, 1963).

         Four groups of 15 male and 15 female rats received 0, 5, 10 and
    20% of caramel colour in their diet for 90 days. No adverse effects
    were noted on appearance, behaviour, survival, body weights, food
    intake, haematology, blood chemistry, urinalysis, organ weights, gross
    and histopathology (Oser, 1963).

         Four groups of 10 male and 10 female rats received 0, 0.015, 0.3
    and 3.0% of caramel colour in their diet for 90 days. No differences
    between test and control animals were noted regarding body weight,
    food consumption, haematology, urinalysis, gross or histopathology
    (Neese, 1964).

         Four groups of rats received 0, 4, 8 and 16% caramel colour in
    their diet for three months. No convulsions or other behaviour
    abnormality or signs of neurological damage were seen. No macroscopic
    or microscopic pathological abnormalities were found in the CNS. Other
    results are still to come (Sharratt, 1971).

    Dog

         Four groups of three male and throe female adult beagles received
    0, 6, 12.5 and 25% of caramel colour in their diet five days per week
    for 90 days. No significant adverse effects were noted due to the test
    compound on growth behaviour, food consumption, mortality, liver
    function, kidney function, haematology, urine analysis, gross and
    histopathology (Kay & Calandra, 1962).

    Long-term studies

    Rat

         Studies on 10 groups of 40 male and 40 female rats are in
    progress extending over two years and using caramels of varying
    4-methylimidazole content at different dietary levels from 2 to 10%
    (CIVO, 1972).

         No formal studies are available but in the course of a
    reproduction study using 25 pairs of 1 male and 1 female rat given 0
    or 0.8 g/kg bw caramel colour in their drinking fluid as part of a
    beverage tested, several pairs survived for two years or longer. No
    deleterious effects on growth were noted. The data are rather
    incomplete (Bachmann et al., 1946).

    Comments:

         A large number of caramel colours has been tested in short-term
    studies in rats and one variety has been tested in dogs. An extensive
    programme of long-term and reproduction studies and embryotoxicity
    including teratogenicity is under way. The acute and short-term
    studies considered reveal that high levels of intake have no adverse
    effects on the CNS. Therefore the acute neurological effects produced
    by high doses of 4-methylimidazole would not appear to be of major
    concern when caramel colours containing small amounts of this
    contaminant are used in food. The analytical evidence suggests the
    presence of 4-methylimidazole in the range of 50-700 ppm in caramel
    colours depending upon the process of manufacture; 200 ppm is taken as
    an average low value for 4-methylimidazole content. Since the effects
    of chronic ingestion of 4-methylimidazole are unknown the above
    mentioned long-term studies as well as more adequate reproduction,
    embryotoxicity including teratology studies have been initiated on
    caramel colours produced by the ammonia-ammonium sulfate process.

    EVALUATION

    Level causing no toxicological effect

         Rat: 20% (200 000 ppm) in the diet equivalent to 10 000 mg/kg bw.

    Estimate of acceptable daily intake for man

         0-100 mg/kg bw*,**

    FURTHER WORK OR INFORMATION

         Required before June 1978

         Results of long-term and reproduction studies on caramel colours
    prepared by the ammonia or ammonium sulfate process containing several
    levels of 4-methylimidazole.

              

    *    Temporary.

    **   Based on a product having a colour intensity of 20 000 EBC units,
    containing not more than 200 ppm of 4-methylimidazole.

    REFERENCES

    Battelle Memorial Institute (1971) Unpublished report dated 4 May 1971

    Bachmann, G. et al. (1946) J. Nutr., 32, 85

    Chacharonis, P. (1960) Unpublished report No. S.A. 54219 of Scientific
         Associates Inc.

    Chacharonis, P. (1963) Unpublished report No. S.A. 79105 of Scientific
         Associates Inc.

    CIVO, 1972, Unpublished report submitted to WHO

    Foote, W. L., Robinson, R. F. & Davidson, R. S. (1958) Unpublished
         report of Battelle Memorial Institute

    Haldi, J. & Wynn, W. (1951) Unpublished report of Emory University

    Haldi, J. (1958) Unpublished report of Emory University

    Heyns, K. (1970) Unpublished report of Technical Caramel Committee

    Heyns, K. (1971) Unpublished summary report

    Kay, J. H. & Calandra, J. C. (1962) Unpublished report of Industrial
         Bio-test Laboratories Inc.

    Komoto, M. (1962) J. Agric. Chem., 36, 305

    Neese, P.O. (1964) Unpublished report of Wisconsin Alumni Research
         Foundation

    Nishie, K., Waiss, A. C. & Keyl, A. C. (1969) Toxicol. Appl.
         Pharmacol., 14, 301

    Nishie, K., Waiss, A. C. & Keyl, A. C. (1971) Toxicol. Appl.
         Pharmacol., 17, 1

    Oser, B. L. (1963) Unpublished report No. 83911 of Food and Drug
         Research Laboratories

    Prier, R. F. (1960) Unpublished report No. 9070599 of Wisconsin Alumni
         Research Foundation

    Sharratt, M. (1971) Unpublished report


    See Also:
       Toxicological Abbreviations