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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.


    ANNATTO EXTRACTS

    Biological Data

    Biochemical aspects

    None available.

    Acute toxicity

                                                                         
    Animal  Route   Extract type     LD50          Reference
                                     (mg/Kg 
                                     body-weight)
                                                                         

    Mouse   i.p.    water soluble    700           Durham & Allard, 1960
    Rat     oral    fat soluble      > 50 ml       Van Esch at al., 1959
    Rat     oral    fat soluble      > 25 ml       Van Esch at al., 1959
    Rat     oral    water soluble    > 35 ml       Van Each at al., 1959
                                                                         

    Administration of an aqueous extract of bixa root depressed
    spontaneous motor activity in the mouse, the intraperitoneal ED being
    21 mg/kg body-weight. The extract also affects the volume of gastric
    secretion but not its pH (400 mg/kg intraduodenally). It has some
    antispasmodic (1 mg/m1 isolated guinea-pig ileum) and hypotensive
    properties (I.V. 50 mg/kg rat) (Durham & Allard, 1960).

    Special tests

    Mutagenic action was tested in a concentration of 0.5 g/100 ml in
    cultures of Escherichia coli. No mutagenic effect was found (Luck &
    Rickerl, 1960).

    Short-term studies

    Mouse. Seventy male and 30 female mice were injected s.c. with 0.1
    ml annatto. Occasionally a sarcoma was produced at the site of
    injection. No definite effect was seen on distant tumoric development
    either as regards time of appearance or number, (Engelbreth - Holm &
    Inverson, 1955). Fifty male and 50 female mice were painted twice a
    week for three months at the interscapular region with 0.05 ml 50 per
    cent. annatto in benzene. No skin papillomas or other tumours were
    encountered (Engelbreth - Holm & Inverson, 1955).

    Rat. Three groups of 10 males and 10 female rats were fed 0 per
    cent. and 2 per cent. of fat soluble annatto and 2 per cent. water
    soluble annatto for 13 weeks. Food intake, growth, haematological
    examination, organ weights and histopathology of major organs showed
    no abnormalities (van Esch et al., 1959). Two groups of 10 male and 10
    female rats were given 0 and 1000 mg/kg body-weight, of annatto orally
    for 100 days. No abnormalities were seen (Zbinden & Studer, 1958). Two

    groups of 10 male and 10 female rats ware injected s.c. at the same
    site for 36 weeks, three times per week, with 0.05 ml corn oil and fat
    soluble annatto. After observation for 24 months there were no local
    tumours (van Esch et al., 1959).

    Dog. Two groups of 3 male and 3 female beagles were fed 0 or 2.7 per 
    cent. on the diet of fat soluble extract of annatto seed for nine
    weeks, then fed normal diet for five weeks and then fed only 1.35 per
    cent. in the diet of fat soluble extract in capsules for 38 weeks. No
    abnormalities were found as regards growth, food intake, mortality,
    liver and kidney function, haematology or histopathology. One female
    dog died in the test group. The liver of this animal showed
    hepatocellular degeneration (Nay & Calandra 1961a). Four groups of 3
    male and 3 female beagles were fed in their diet 0, 5 per cent. and 10
    per cent. aqueous extract of annatto seed for one year. The fourth
    group received 20 per cent. aqueous extract for 16 weeks in their diet
    and then half of the extract in the diet and half in gelatine capsules
    for 36 weeks. Controls received 0.48 per cent. potassium chloride.
    Growth inhibition and reduced food intake occurred at the 20 per cent.
    dietary level. Mortality rate, liver and kidney function tests,
    haematology and histopathology of all major tissues showed no
    abnormalities attributable to the test substance (Kay & Calandra
    1961b).

    Pig. Three groups of 2 male and 1 female pig were fed 0 per cent.
    and 1 per cent. fat soluble annatto and 1 per cent. water soluble
    annatto for 21 weeks. One animal in the test group died from a cause
    unrelated to the test substance. Food intake, growth, haematology,
    organ weights and histopathology of all major tissues was normal (van
    Esch et al., 1959).

    Long-term studies

    Mouse. Fifty female and 50 male mice were fed daily, one 
    drop of a 10 per cent. solution of annatto in soy oil for 24 months.
    There was no significant difference from a similar control group on
    normal diet (Engelbreth - Holm & Iverson, 1955). Two groups of 50 male
    and 50 female mice were fed either 0.5 per cent. corn oil or 0.5 per
    cent. fat soluble annatto for their life span. The same animals also
    received s.c. 0.1 ml oil three times per week for 17 months. Two other
    groups of 25 males and 24 females were fed for their life span 0 per
    cent. or 0.05 per cent. concentrated fat soluble annatto and the same
    animals were also injected 0.001 ml s.c. for 10-1/2 months. Cyst
    formation with local necrosis was seen at the site of injection. Most
    animals died between 15 and 21 months due to intercurrent infection.
    No statistically significant increase in tumour production was
    observed (van Esch et al., 1959).

    Rat. Two groups of 100 female rats were given daily 26 mg annatto in
    soy oil for 26 months. No effect was noted on the pathological
    experience of the two groups (Engelbreth - Holm & Iverson, 1955).
    Three groups of 10 male and 10 female rats received corn oil with 0,
    0.5 per cent. fat soluble erratic and 0.5 per cent. water soluble

    annatto for their life span. These extracts varied in total bixin
    content from 0.2-2.6 per cent. Two daughter generations were bred each
    being fed similar diets for 7 and 8-1/2 months. No deleterious effect
    was observed on growth and reproduction. No teratogenic effects were
    seen. No consistent effect on mortality was noted in the three
    generations. Organ weights and tumoric incidence were comparable in
    all groups (van Each et al., 1959). Two groups of 10 male and 10
    female rats were fed 0 or 0.05 per cent. of concentrated fat soluble
    annatto for 32 months. A first filial generation received the same
    diet for seven months. No deleterious effects were seen on growth and
    reproduction, mortality, organ weights and tumoric production (van
    Esch et al., 1959).

    Comments

    Adequate long-term tests in two species have been performed on a well
    defined type of extract containing 0.2 - 2.6 per cent. of carotenoids
    expressed as bixin. In addition, the absence of tumorigenic activity
    on subcutaneous injection or after painting the skin of mice has been
    shown, No data are available on the metabolism of this material or any
    of its major components. Although the contents of bixin and norbixin
    in the extracts tested are known the data do not permit evaluation of
    these pure components. Investigation of the storage of the lipid
    soluble extract in tissue fats my be useful.

    EVALUATION

    Level causing no toxicological effect in the rat

    0.5 per cent. (= 5000 ppm) in the diet equivalent to 250 mg/kg
    body-weight per day.

    Estimate of acceptable daily intake for man

                           mg/kg body weight

    Temporary acceptance:       0 -1.25

    Further work required by June 1972

    Metabolic studies on the major carotinoids of annatto.

    REFERENCES

    Durham, N. W. & Allard, R. K. (1960) J. Amer. pharm. Assoc. 49, 218

    Engelbreth - Holm, J. & Iverson, S. (1955) Acts. Path. Microb. Scand
    37, 483

    van Esch, G. J., van Genderen, H. & Vink, H. H. (1959) Z. Lebensm -
    Unters.,111, 93

    Kay, J. H. & Calandra, J. C. (1961a) Unpublished report by Industrial
    BIO-TEST Laboratories Inc., 25/7/61 to Marachall Dairy Lab. Inc.

    Kay, J. H. & Calandra, J. C. (1961b) Unpublished report by Industrial
    BIO-TEST Laboratories Inc., 6/3/61 to Marschall Dairy Lab. Inc.

    Luck, H. & Rickerl, E. (1960) Z. Lebensm -Unters., 112, 157

    Zbinden, G. & Studer, A. (1958) Z. Lebensm - Unters., 108, 113
    


    See Also:
       Toxicological Abbreviations
       Annatto Extracts (WHO Food Additives Series 52)
       Annatto extracts (WHO Food Additives Series 6)
       Annatto extracts (WHO Food Additives Series 17)
       Annatto extracts (WHO Food Additives Series 44)
       ANNATTO EXTRACTS (JECFA Evaluation)