These compounds were previously evaluated for acceptable daily
    intake (ADI) by the Joint FAO/WHO Expert Committee on Food Additives
    in 1964, 1969 and 1974 (see Annex I, Refs. 8, 19 and 34).

         Toxicological monographs were issued in 1969 and 1974 (see Annex
    I, Refs. 20 and 35).

         Since the previous reviews, further data have become available
    and are incorporated into this revised monograph.



    Special studies on the accumulation and excretion

         The metabolism of 3 annatto preparations has been studied. The
    materials were:

    (1) OSB - a vegetable oil solution which contains bixin and the
    thermal degradation pigment;

    (2) R10- a vegetable oil suspension which contains mainly bixin;

    (3) WSA - a water-soluble preparation which contains mainly norbixin.

         These annatto preparations are similar, respectively, to Annatto
    FL, Annatto FL10, and Annatto WL, which were used by van Esch et al.
    (1959) in their long-term feeding tests.

         Groups of 4 male and 4 female rats were fed 0 and 5% of OSB, R10,
    and WSA annatto extracts for a 4-week period. Rats were fed annatto
    extracts in the diet for the first 2 weeks only or the last 2 weeks
    prior to the killing.

         Direct evidence for the intestinal absorption of annatto pigments
    is provided by the presence of these pigments in blood (serum) in the
    same proportion as they are to be found in the commercial products.
    This is consistent with the finding that the pigments excreted in
    faeces were also in the same proportions as they are to be found in
    the commercial products. TLC of the sera of rats fed OSB, R10 and WSA
    also revealed components which are possibly metabolites of annatto

         There is no evidence that the major components of annatto
    extracts colour the adipose tissue of rats. The fact that WSA-fed rats
    do not have coloured fat also suggests that the major annatto pigments
    are not involved in the coloration.

         The yellow pigment which colours the adipose tissue of rats fed
    OSB and R10 is possibly identical to a minor component which occurs in
    OSB and R10. The marked difference in the coloration of adipose tissue
    from rats fed OSB or R10 in weeks 3 and 4 from those fed during weeks
    1 and 2 indicates that the adipose tissue store of pigment is rapidly
    depleted when ingestion of annatto ceases.

         These findings confirm and carry further the previous
    observations of van Esch (1959) (Philp, 1981).

         Five groups of 4 male and 4 female rats were given by stomach
    tube a single dose of undiluted WSA (10 ml/kg), R10 (2 ml/kg), OSB
    (2 ml/kg) annatto, sunflower oil (2 ml/kg) and water (10 ml/kg).

         The annatto pigments in rat's blood were determined 3 hours and
    24 hours after treatment.

         In rats dosed with WSA, the blood level was 270 mg/100 ml after
    3 hours and 10 mg/100 ml after 24 hours. The results suggest that
    within 24 hours WSA was almost completely absorbed and metabolized.

         In rats dosed with OSB, the blood level was 62 mg/100 ml after
    3 hours and 19 mg/100 ml after 24 hours. Thus, OSB was metabolized
    less rapidly than WSA, presumably because the OSB pigments would need
    to be metabolized to water-soluble pigments, similar to those of WSA,
    before being metabolized further. Likewise, R10 was metabolized
    more slowly than WSA. The relatively low blood levels of annatto
    preparation 24 hours after large, oral doses provides evidence of the
    ability of the rat to metabolize both water-soluble and fat-soluble
    annatto pigments (Philp, 1981).


    Special studies on mutagenicity

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

         Annatto extract (bixin content not specified) was investigated
    for ability to induce DNA damage in an E. coli rec-assay. Induction
    of reverse mutations in E. coli trp vvr A and Salmonella
    typhimurium TA 1538 his rfa uvrB was investigated in fluctuation
    assays. Both types of assays were conducted with and without metabolic

    activation using caecal extracts and liver microsomes from rats.
    Annatto extract did not induce detectable genotoxicity in these test
    systems (Haveland-Smith, 1981).

    Acute toxicity

    Animal   Route   Extract type    (mg/kg bw)   Reference

    Mouse    i.p.    Water soluble     700        Durham & Allard, 1960

    Rat      Oral    Fat soluble       >50 ml     van Esch et al., 1959
             Oral    Fat soluble       >25 ml     van Esch et al., 1959
             Oral    Water soluble     >35 ml     van Esch et al., 1959

         Administration of an aqueous extract of bixa root depressed
    spontaneous motor activity in the mouse, the intraperitoneal LD50
    being 21 mg/kg bw. The extract also affects the volume of gastric
    secretion but not its pH (400 mg/kg intraduodenally). It has some
    antispasmodic (1 mg/ml isolated guinea-pig ileum) and hypotensive
    properties (i.v. 50 mg/kg rat) (Durham & Allard, 1960).

    Short-term studies


         Seventy male and 30 female mice were injected s.c. with 0.1 ml
    annatto. Occasionally a sarcome 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 & Iversen,
    1955). Fifty male and 50 female mice were painted twice a week for
    3 months at the interscapular region with 0.05 ml 50% annatto in
    benzene. No skin papillomas or other tumours were encountered
    (Engelbreth-Holm & Iversen, 1955).


         Three groups of 10 male and 10 female rats were fed 0 and 2% of
    fat-soluble annatto and 2% 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 bw of annatto orally for 100 days. No abnormalities were
    seen (Zbinden & Studer, 1958).

         Two groups of 10 male and 10 female rats were injected s.c. at
    the same site for 36 weeks, 3 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).

         Four groups of 6 male and 6 female rats were fed 0% purified
    diet, 0.1% OSB; 0.02% R10, 0.1% WSA of annatto for 1 year. Another
    group received the purified diet containing 0.1% OSB, 0.02% R10, and
    0.1% WSA. Body weights and food intakes reveal that rats fed diets
    containing annatto preparations consumed as much food as rats in the
    control group fed purified diet and that in all groups the body
    weights were similar throughout the 52 weeks. The liver, kidney, and
    adipose tissue weights show that the animals were in good health at
    the end of the year. No significant difference was observed between
    animals on the various diets. Analysis of the extracts of liver,
    kidneys, adipose tissue and carcass of rats fed diets containing 1 or
    all of the annatto preparations failed to reveal the presence of any
    of the annatto pigments. Major carotenoids do not accumulate in body
    tissues when extracts are fed for a year and the minor component also
    does not accumulate (Philp, 1981).


         Two groups of 3 male and 3 female beagles were fed 0 or 2.7% in
    the diet of fat-soluble extract of annatto seed for 9 weeks, then fed
    normal diet for 5 weeks and then fed only 1.35% 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 (Kay &
    Calandra, 1961a).

         Four groups of 3 male and 3 female beagles were fed in their diet
    0%, 5% and 10% aqueous extract of annatto seed for 1 year. The fourth
    group received 20% 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% potassium chloride. Growth inhibition
    and reduced food intake occurred at the 20% 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).


         Three groups of 2 male and 1 female pigs were fed 0 and 1% fat-
    soluble annatto and 1% 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 were normal (van Esch et al., 1959).

    Long-term studies


         Fifty male and 50 female mice were fed daily 1 drop of a 10%
    solution of annatto in soy oil for 24 months. There was no significant
    difference from a similar control group on normal diet (Engelbreth-
    Holm & Iversen, 1955).

         Two groups of 50 male and 50 female mice were fed either 0.5%
    corn oil or 0.5% fat-soluble annatto for their life span. The same
    animals also received s.c. 0.1 ml oil 3 times per week for 17 months.
    Two other groups of 25 male and 24 female mice were fed for their life
    span 0 or 0.05% 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).


         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 2 groups (Engelbreth-Holm & Iversen, 1955).

         Three groups of 10 male and 10 female rats received corn oil with
    0, 0.05% fat-soluble annatto and 0.5% water-soluble annatto for their
    life span. Those extracts varied in total bixin content from 0.2 to
    2.6%. 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 3 generations. Organ weights and
    tumoric incidence were comparable in all groups (van Esch et al.,

         Two groups of 10 male and 10 female rats were fed 0 or 0.05% of
    concentrated fat-soluble annatto for 32 months. A first filial
    generation received the same diet for 7 months. No deleterious effects
    were seen on growth and reproduction, mortality, organ weights and
    tumoric production (van Esch et al., 1959).


         In a group of 61 consecutive patients suffering from chronic
    urticaria and/or angioneurotic oedema, 56 patients were provoked
    orally with 25 µl of annatto extract (bixin content 0.065%) during
    elimination diet. Fifteen patients (26%) reacted with a flare-up of
    symptoms (Mikkelsen et al., 1978).

         Single oral doses of OSB (7 mg/kg), R10 (7 mg/kg) and WSA
    (14 mg/kg) were given to adult males and the blood and excreta were
    analysed for annatto pigments. Blood samples were taken between
    2-12 hours after treatment, urine was collected during 7 hours after
    the dose and faeces over the 2 days following the day of treatment.
    WSA (14 mg/kg) produced a blood level of 12 µg/ml after 2-1/4 hours
    which corresponds to 6% of the dose. OSB (7 mg/kg) produced a blood
    level of 2.4 µg/ml after 3 hours which corresponds to 2.4% of the
    dose. R10 (7 mg/kg) produced a blood level of 0.44 µl/ml after 3-1/4
    hours which corresponds to 0.32% of the dose. Blood levels had
    returned to zero 6 hours after WSA (14 mg/kg), OSB (7 mg/kg) and R10
    (7 mg/kg) respectively.

         No annatto pigments were detected in the urine samples and none
    were detected in faeces samples collected the next day. The faeces
    collected the second day after treatment contained 0.17 mg R10
    (0.03% of the dose) and 0.44 mg WSA (0.06% of the dose) but no
    pigments associated with the consumption of OSB were detected. Thus,
    as in the rat, the annatto pigments were absorbed and rapidly cleared
    from the blood (Philp, 1981).

         During 1974-1978 a number of patients with recurrent urticaria
    was orally provoked with up to 10 mg annatto extract (bixin content
    not stated). Of 112 patients tested, 10% reacted to annatto extract
    (Juhlin, 1981).


         Adequate long-term tests in 2 species have been performed on a
    well-defined type of extract containing 0.2-2.6% of carotenoid
    expressed as bixin. Short-term tests in 2 other species suggest lack
    of cumulative action even at levels of 15% carotenoid in vegetable oil
    or 10% in water. The long-term study in the rat provides a basis for

         The metabolic studies requested in 1974 have become available.
    The dog studies indicate that the high levels of carotenoids do not
    produce adverse effects. When lower dietary levels of OSB, R10 and WSA
    were fed to rats for 1 year, no evidence of accumulation of annatto
    pigments was found. Both in the rat and in man, annatto pigments are
    absorbed from the intestine into the blood and clearance from the
    blood is quite rapid.

         It can be concluded that the main carotenoids of annatto are well
    absorbed in both rat and man and do not appear in tissue fat in the


    Level causing no toxicological effect

    Rat: 0.5% (= 5000 ppm) in the diet equivalent to 250 mg/kg bw.

    Estimate of acceptable daily intake for man

    0-0.065 mg/kg bw (expressed as bixin).


    Durham, N. W. & Allard, R. K. (1960) A preliminary pharmacologic
         investigation of the roots of Bixa orellana, J. Amer. pharm.
         Assoc., 49, 218-219

    Engelbreth-Holm, J. & Iversen, S. (1955) Is vegetable annatto butter
         colour carcinogenic? Acta Path. Microb. Scand., 37, 483-491

    van Esch, G. J., van Genderen, H. & Vink, H. H. (1959) Uber die
         chronische Verträglichkeit von Annattofarbstoff, Z. Lebensm.
         - Untersuch., 111, 93-108

    Haveland-Smith, R. B. (1981) Evaluation of the genotoxicity of some
         natural food colours using bacterial assays, Mutation Res.,
         91, 285-290

    Juhlin, L. (1981) Recurrent urticaria: clinical investigation of 330
         patients, Brit. J. Dermatol., 104, 369-381

    Kay, J. H. & Calandra, J. C. (1961a) Unpublished Report by Industrial
         Bio-Test Laboratories, Inc., 25/7/61 to Marshall Dairy Lab. Inc.

    Kay, J. H. & Calandra, J. C. (1961b) Unpublished Report by Industrial
         Bio-Test Laboratories, Inc., 6/3/61 to Marshall Dairy Lab. Inc.

    Lück, H. & Rickerl, E. (1960) Lebensmittelzusatzstoffe und Mutagene
         Wirkung. VI Mitteilung. Prüfung der in Westdeutschland
         zugelarsenen und ursprünglich vorgeschlagenen Lebensmittel-
         farbstoffe auf Mutagene Wirkung an E. coli, Z. Lebensm.
         -Untersuch., 112, 157-174

    Mikkelsen, H. et al. (1978) Hypersensitivity reactions to food colours
         with special reference to the natural colour Annatto extract
         (butter colour), Arch. Toxicol., Suppl. 1, pp. 141-143

    Philp, J. (1981) Unpublished report by Unilever Research Division to

    Zbinden, G. & Studer, A. (1958) Tierexperimentelle Untersuchungen Uber
         die chronische Verträglichkeit von ß-Caratin, Lycopin,
         7,7-Dihydro-ß-carotin und bixin, Z. Lebensm.-Untersuch., 108,

    See Also:
       Toxicological Abbreviations
       Annatto Extracts (WHO Food Additives Series 52)
       Annatto extracts (FAO Nutrition Meetings Report Series 46a)
       Annatto extracts (WHO Food Additives Series 6)
       Annatto extracts (WHO Food Additives Series 44)
       ANNATTO EXTRACTS (JECFA Evaluation)