INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF SOME
FOOD COLOURS, EMULSIFIERS, STABILIZERS,
ANTI-CAKING AGENTS AND CERTAIN
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.
Animal Route Extract type LD50 Reference
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).
Mutagenic action was tested in a concentration of 0.5 g/100 ml in
cultures of Escherichia coli. No mutagenic effect was found (Luck &
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
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).
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).
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.
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.
Durham, N. W. & Allard, R. K. (1960) J. Amer. pharm. Assoc. 49, 218
Engelbreth - Holm, J. & Iverson, S. (1955) Acts. Path. Microb. Scand
van Esch, G. J., van Genderen, H. & Vink, H. H. (1959) Z. Lebensm -
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