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.
These compounds have been evaluated for acceptable daily intake
by the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
Ref. No. 20) in 1969.
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.
No information available.
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 &
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
Rat oral fat soluble > 25 ml Van Esch et al., 1959
Rat 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 ED50
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 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 & Iverson,
1955). Fifty male and 50 female mice were painted twice a week for
three months at the interscapular region with 0.05 ml 50% annatto in
benzene. No skin papillomas or other tumours were encountered
(Engelbreth-Holm & Iverson, 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, 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).
Two groups of three male and three female beagles were fed 0 or
2.7% 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% 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
three male and three female beagles were fed in their diet 0, 5% and
10% aqueous extract of annatto seed for one 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 two male and one 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.,
Fifty female and 50 male mice were fed daily, one 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 & Iverson, 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 three times per
week for 17 months. Two other groups of 25 males and 24 females 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 inter-
current 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 two groups (Engelbreth-Holm & Iverson, 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 0.2-2.6%. Two
daughter generations were bred each being fed similar diets for seven
and eight-and-a-half 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
Esch et al., 1959). 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 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% of carotenoid
expressed as bixin. Short-term tests in two other species suggest a
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 evaluation. However, information on the metabolism is
lacking while the dog studies indicate that the high levels of
carotenoids do not produce adverse effects. Studies on the metabolism
of the carotenoids are in progress.
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
0-1.25* mg/kg bw**
FURTHER WORK OR INFORMATION
Required by June 1978.
Completion of metabolic studies presently in progress.
Durham, N. W. & Allard, R. K. (1960) J. Amer. pharm. Assoc., 49, 218
Engelbreth-Holm, J. & Iverson, S. (1955) Acta. Path. Microb. Scand.,
van Esch, G. J., van Genderen, H. & Vink, H. H. (1959) Z. Lebensm.
Untersuch., 111, 93
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) Z. Lebensm.-Untersuch., 112, 157
Zbinden, G. & Studer, A. (1958) Z. Lebensm.-Untersuch., 108, 113
** Expressed as bixin.