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.
Microchemical methods have shown that filterable indigotine in the
blood plasma of rabbits is excreted through the glomeruli. The
filterable fraction is about 15 per cent. while 75 per cent. of the
colour is excreted by the tubules. The lumina of the tubules and
Bowman's capsules were injected with solutions of indigotine. Ten mg
per cent. was the lowest concentration which left detectable traces of
the colour in the kidneys. When an increased dose of colour is
injected and the interval between the injection and the excision
of the kidney shortened, the colour can, be detected in the
intracapsular spaces. The tubular excretion of the indigotine
resembles quantitatively that of phenol red (Kempton et al., 1937).
After intravenous injection of 35S-labelled dye in the rats, 63 per
cent. of the radioactivity appeared in the urine in six hours and 10
per cent. in the bile. The metabolites isatin-5-sulfuric acid and
5-sulfoanthranilic acid appeared in the urine after two hours. After
oral administration, only three per cent. of the radioactivity
appeared in urine in three days, suggesting poor absorption from the
alimentary tract. Faeces contained 60-80 per cent.of the oral dose.
The faecal content was due to lack of absorption, not biliary
excretion (Lethco & Webb, 1966).
Animal Route LD50 Reference
Mice Oral >2 500 US FDA, 1969
Mice Subcutaneous 405 US FDA, 1949
Rat Oral 2 000 Lu & Lavallée, 1964
Rats Intravenous 93 US FDA, 1969
Five rats were given subcutaneous twice daily injections for three
days and killed on the fourth day. An aqueous solution at a level of
250 mg/kg body-weight was used. No oestrogenic activity (normal
uterine weight) was detected but the animals lost weight (Graham &
In experiments with guinea-pigs it was found that this colour had no
sensitization activity (Bär & Griepentrog, 1960).
The colour was tested for mutagenic effect in a concentration of 0.5
g/1 0.5 g/100 ml in cultures of Escherichia coli. No mutagenic
effect was found. (Lück & Rickerl, 1960).
Isatin-5-sulphonic acid, one of the metabolites of the colour, was fed
to groups of three-week-old rats, 10 male and 10 females per group, at
levels of 0, 0.25, 0.5, 1 and 2 per cent. for a period of 13 weeks
followed by gross and histopathology examinations. The "no-effect"
level of the compound was considered to be two per cent. (FDA, 1969).
Rat. Twenty rats were given first 1 ml two per cent. solution
subcutaneous later only 0.5 ml of a 0.5 per cent. solution. Fifty-five
injections were administered over a period of seven months. Control
groups received injections of 50 per cent. glucose or 0.9 per cent.
sodium chloride. All animals were observed for lifespan. No local
tumours and only one auxiliary tumour in the test group were found
(Oettel et al., 1965).
Dog. Two groups, consisting of two male and two female beagles were
given 1.0 per cent. and two per cent. Indigotine in the diet for a
two-year feeding trial. One male and one female served as controls.
Two dogs on the high level died at 19 weeks; they were replaced and
another dog was added to the control group. Two more on the high level
died, at 21 and 40 weeks; one on the low level died at 36 weeks; and
one control died at 34 weeks. Deaths were due to virus infections.
There were no clinical signs, gross lesions or microscopic pathology
attributable to the dye (Hansen et al., 1966).
Mouse. Groups of 25 male and 25 female mice received weekly for 104
weeks subcutaneous injections of 2.5 mg indigotine as a one per cent.
aqueous solution, the control group of 50 receiving 0.25 ml
physiological saline. Many mice died from acute convulsions
immediately after injection of the test dye but otherwise no
deleterious effects attributable to the subcutaneous injections were
noted. Tumours were randomly distributed among test and control groups
(Hansen et al., 1966).
Rat. A group of 20 male and 20 female rats received the dye as one
per cent. solution for two years. Twenty males and 20 females were
No adverse effects were observed on growth, reproduction and survival
and no specific gross or microscopic pathology was noted (organs not
stated). There was no difference in tumour incidence between the
groups (Oettel et al., 1965).
Groups of 24 rats, equally divided by sex, were fed the colour at 0,
0.5, 1.0, 2.0 and 5.0 per cent. for two years. The growth of males was
significantly inhibited at 2.0 and 5.0 per cent. There was no change
in mortality, organ weights or haematology, nor any gross or
microscopic pathology related to treatment (Hansen et al., 1966).
Groups of 80 and 100 rats were injected weekly with a two per cent.
aqueous solution or an equivalent volume of saline solution for two
years. Survival of test animals did not differ from controls. Fourteen
of 80 injected rats had a fibrosarcoma at the site of injection and
one saline injected rat developed a fibroma at the injection site. No
other effects were observed (Hansen et al., 1966).
The production of a high percentage of local sarcomata at the site of
subcutaneous injections in rats led in the past to considerable
discussion and consequently to extensive studies on this colour and
the special condition of the experiment and does not constitute
evidence of carcinogenicity by the oral route.
The metabolic studies on this colour are fairly complete and the two
long-term studies in the rat do not point to any significant toxic
effects. A thirteen-week study on the major metabolite revealed no
toxic effects. Human metabolic studies would be useful.
Level causing no toxicological effect in the rat
One per cent. (= 10 000 ppm) in the diet equivalent to 500 mg/kg body
Estimate of acceptable daily intake for man
Temporary acceptance 0 -2.5
Further work required by June 1974
Two-year study in a non-rodent mammalian species
Bär, F. & Griepentrog, F. (1960) Med. u. Ernähr., 1, 99
Graham, R. C. B. & Allmark, M. G. (1959) Toxicol. appl. Pharmacol.,
Hansen. W. H., Fitzhugh, O. G., Nelson, A. A. & Davis, K. J. (1966)
Toxicol. appl. Pharmacol., 8, 29
Kempton, R. T., Bott, P. A. & Richards, A. N. (1937) Amer. J. Anat.,
Lethco, E. J. & Webb, J. M. (1966) J. Pharmacol. exp. Ther., 154,
Lu, F. C. & Lavallée, A. (1964) Canad. pharm. J., 97, 30
Lück, H. & Rickerl, E. (1960) Z. Lebensmitt.-Untersuch., 112, 157
Oettel, H., Frohberg, H., Nothdurft, H. & Wilhelm, G. (1965) Arch.
für Toxikol., 21, 9
United States Food and Drug Administration (1969) Unpublished report