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.
The metabolic behaviour and excretory pattern for erythosine have been
studied in adult rats. The colour was given by stomach tube in
log-spaced doses from 0.5-500 mg per kg body weight. In five days the
recovery in the excreta was 102 per cent. After an intravenous
application of 3 mg per kg body weight the urine and bile for the
initial two to four hours was collected, an average of 55 per cent
(50.4-58.0 per cent.) of the administered quantity was found in the
bile. In the urine 1.3 per cent. (0.8-1.8 per cent.). No glucuronic
acid conjugation is found.
This colour was found to be largely excreted in the faeces by rats
(55-72 per cent.) and despite the presence of two groups capable of
undergoing conjugations, no colour could be identified in the urine. A
small amount of the colour (0.4-1.7 per cent.) was excreted in the
bile (Daniel, 1962).
Consideration has been given to the possibility that iodide may be
liberated from erythrosine and may disturb thyroid function. In the
rat, erythrosine is metabolically stable and 100 per cent. of the
amount is ingested and excreted with its iodine content intact after
administration of 500 mg/kg (Webb et al., 1962). Protein-bound and
total blood iodine levels were elevated in rats given erythrosine by
stomach tube twice weekly in a chronic study (Bowie et al., 1966).
However the elevated PBIs (protein-bound iodine) were due to
interference by erythrosine in PBI determinations rather than thyroid
disfunction in rats and gerbils (USFDA, 1969). In man, oral
administration of 16 mg of erythrosine daily for 10 days resulted in
an increase of protein-bound iodine in the serum from 6-11 µg/100 ml
after 15-20 days, followed by a sharp decline in iodine levels in the
next 10 days with gradual return to the initial value in three months
(Andersen et al., 1964).
Large doses of erythrosine labelled with I131 given orally to rats
inhibited uptake of I131 by the thyroid of treated animals. Daily
doses over 1 mg are necessary for this effect (Marignan et al., 1965).
When cherries coloured with erythrosine are stored in plain cans,
fluorescein is readily formed by interaction of the tin-iron couple
present. This does not occur in lacquered cans. The production of
fluorescein (with 4 I atoms) from erythrosine occurs in presence of
metallic iron and/or tin and free organic acid (result of
electro-chemical reduction in the can) (Dickinson & Raven, 1962).
It was found that this colour in a concentration of 200-400 mg/litre,
inhibited the action of pepsin but had no effect on lipase activity
(Diemair & Hausser, 1951).
It was also found that this colour had in vivo as well as in vitro
haemolytic effect. In the in vivo studies the mouse was used
Erythrosine was administered to rats in doses of 5, 10, 15 and 50 mg
per rat weighing 200-250 g, twice weekly for six months. Haemoglobin
was reduced at three months, also the red cell count. The cholesterol
levels of males were depressed. Excretion of the dye was mainly in the
faeces and predominantly unchanged (Bowie et al., 1966).
This colour was tested for mutagenic activity and showed a very slight
but statistically significant mutagenic effect on Escherichia coli
in concentrations of 0.5 g/100 ml. It was found that xanthene molecule
itself was the causative factor and that the substituent groups only
modify the effect (Lück et al., 1963; Lück & Rickerl, 1960).
Animal Route LD50 Reference
mg/kg body weight
Mouse I.v. 370 Waliszewski, 1952
I.p. 300 DFG, 1957
Rat Oral >2 000 DFG, 1957
Lu & Lavallée, 1964
Rabbit I.v. 200 DPG, 1957
Gerbil Oral 1 930 USFDA, 1969
A group of five young rats were given subcutaneous injections twice
daily for three days. The rats were killed on the fourth day. The
colour was administered in aqueous solution at a level of 250 mg per
kg body weight each injection. No oestrogenic activity (normal uterine
weight) was detected (Graham & Allmark, 1959).
Guinea-pig: In experiments with guinea-pigs it was found that this
colour had no sensitization activity (Bar & Griepentrog, 1960).
Rat: 1 ml of a 0.8 per cent. solution, when injected subcutaneously
twice weekly did not produce any changes likely to lead to sarcomata
formation (Grasso & Golberg, 1966).
Dog: Two-year feeding studies were conducted with three male and
three female beagles at levels of 0, 0.5, 1.0 and 2.0 per cent. All
dogs survived the study. No gross or microscopic pathology related to
the colour was seen (USFDA, 1969).
Mouse: A total of 122 male and female mice produced by mixed
breeding from five different strains were given a diet containing 1 mg
per animal per day of the colour. Mice at the age of 50-100 days were
used. A number of the animals were sacrificed after an observation
period of 500 days and the survivors of the rest after 700 days. A
total of 168 mice was used as control. Positive control groups which
were given O-aminoazotoluene and dimethylaminoasobenzene were also
included. In these groups the formation of liver tumours was noted
after approximately 200 days. The incidence of tumours in mice
receiving the colour was not significantly greater than in the
controls (Waterman & Lignac, 1958).
Chronic feeding studies were conducted with mice. Seventy mice were
fed at one and two per cent. Because of the small number of animals
surviving the experiment and the small number of tumours found, no
effect of tumour formation could be attributed to the colour (USFDA,
Rat: Groups of 24 weanling rats, evenly divided by sex, were fed
this colour at 0, 0.5, 1.0, 2.0 and 5.0 for two years. Slight growth
depression was observed in the animals at the five per cent. level,
and those above 0.5 per cent. had distended caecums but
microscopically the distended caecums showed normal histology. The
statistical evaluation of the rat study revealed no changes in organ
weights at the highest level. There was some diarrhoea at the five per
cent. level. There was no difference in survival (USFDA, 1964). Forty
rats were fed one per cent. of the colour for two years. Six animals
died between time. No tumours were found (DFG, 1957).
The colour was fed at a level of four per cent. of the diet to five
male and five female rats for periods up to 18 months. Gross staining
was observed in the glandular stomach and small intestine and granular
deposits in the stomach, small intestine and colon. Hepatic cirrhosis
was noted in one case out of four rats living up to 12 months. Fifty
control animals observed for 20 months or more failed to develop
tumours, or hepatic cirrhosis (Willheim & Ivy, 1953).
Groups of 100-day-old rats, 50/test level and 100/control, evenly
divided by sex, were fed 0, 0.5, 1.0, 2.0 and 4.0 per cent.
erythrosine for 24 months. Groups of 15 male and 15 female 100-day-old
rats were intubated twice a week for 19 months with erythrosine at 0,
100, 235, 750 and 1500 mg/kg. Body weight decreases were seen at two
and four per cent. Elevated PBIs, due to interference by erythrosine
in PSI determination rather than thyroid dysfunction, were seen. There
were no other haematological differences. No adverse gross pathology
was noted; histopathology has not been completed (USFDA, 1969).
Feeding with one per cent, of the colour for two years did not
influence the body weight or fertility of Wistar rats. There was no
pathological damage and no significant difference in tumour incidence
from control rats (Oettel et al., 1965).
Twenty rats were subjected to weekly subcutaneous injections of 1 ml
of a five per cent. aqueous solution for 596 days. The total quantity
of colour administered was 2.65 g/animal. Seven rats survived 300 days
or more. No tumours were observed (Umeda, 1956).
Eighteen rats were injected subcutaneously 1 ml, of a two per cent. or
three per cent. solution per week for periods of 94-99 weeks. No
tumours either at the site of the injections or in the other parts of
the body were observed (Nelson & Hagan, 1953).
Gerbils: Groups of gerbils, 30/test level and 60/control, evenly
divided by sex, were fed erythrosine at 0, 1.0, 2.0 and 4.0 per cent.
for 24 months. Groups of gerbils, 40/test level and 60/control, evenly
divided by sex, were intubated twice a week for 19 months with 0, 200,
750 and 900 mg/kg (those on 900 mg received 1200 mg for the first
three months). Body-weight decreases were seen at all feeding levels
(only females at one per cent.). Elevated PBIs, due to interference by
erythrosine with PSI determination, were seen. No other haematological
differences were seen. No adverse gross pathology was noted.
Histopathology has not been completed (USFDA, 1969).
The long-term studies in two species are adequate. Most of the colour
is excreted in the faeces and some of the absorbed colour is excreted
via the bile. Elevation of protein-bound iodine levels has been
observed although no toxicological significance in relation to thyroid
activity can be assigned to this observation at present. Conversion to
fluorescein, a nephrotoxic compound is possible and should be avoided.
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
Studies on the metabolism in several species and preferably in man and
elucidation of the mechanism underlying the effect of this colour on
plasmabound iodine levels.
Bär, F. & Griepentrog, F. (1960) Med. u. Ernähr., 1, 99
BIBRA (1967) Fd Cosmet. Toxicol., 5, 109
Bowie, W. C., Wallace, W. C. & Lindstrom, H. V. (1966) Fed. Proc.,
Daniel, J. W. (1962) Toxicol. appl. Pharmacol., 4, 572
Deutsche Forschungsgemeinschaft. Farbstoff Kommission (1957)
Dickinson, D. & Raven, T. W. (1962) J. Sci. Food Agric., 13, 650
Diemair, W. & Hausser. H. (1951) Z. Lebensmitt.-Untersuch., 92, 1965
Graham, R. C. B. & Allmark, M. G. (1959) Toxicol. appl. Pharmacol.,
Grasso, P. & Golberg, L. (1966) Fd. Cosmet. Toxicol., 4, 269
Lu, F. C. & Lavallée, A. (1964) Canad. pharm. J., 97, 30
Lück, H. & Rickerl, E. (1960) Z. Lebensmitt.-Untersuch. 122, 157
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(Basel). 26, 206
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Montpellier, 24, 127
Nelson, A. A. & Hagan, E. C. (1953) Fed. Proc., 12, 397
Umeda, M. (1956) Gann, 47, 51
United States Food and Drug Administration (1969) Unpublished report
Waliszewski, T. (1952) Acta Pol. pharm., 9, 127
Waterman, N. & Lignac, G. O. E., as summarized by Genderen, H. van
(1958) Acta physiol. pharmacol. neerl., 7, 35
Webb, J. M., Fonda, M. & Brouwer, E, A. (1962) J. Pharmacol. exp.
Ther., 137, 141
Willheim, R. & Ivy, A. C. (1953) Gastroenterology, 23, 1