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
(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 &
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).
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).
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
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 &
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).
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).
OBSERVATIONS IN MAN
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
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.,
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,