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.
This compound has been evaluated for acceptable daily intake by
the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
Refs Nos 10 and 20) in 1964 and 1969.
Since the previous evaluation additional data have become
available and are summarized and discussed in the following monograph.
The previously published monographs have been expanded and are
reproduced in their entirety below.
"Ponceau 4 R is reduced in vitro by rat caecal contents
(Walker, 1968), but Ryan and Wright (1961) showed that rats excreted
30-45% of an intravenous dose unchanged in the bile within 6 hours.
The bile was coloured in rats and mice given intraperitoneal doses of
the dye (Gaunt et al., 1967)."
Special studies on mutagenicity
This colour was tested for mutagenic effect in a concentration of
0.5 g/100 ml in cultures of Escherichia coli. No mutagenic effect
was found (Lück & Rickerl, 1960).
Special studies on sensitization
In experiments with guinea-pigs it was found that this colour had
no sensitization (Bär & Griepentrog, 1960). A negative test for Heinz
bodies was obtained after administering this colour in a 5% aqueous
solution by stomach tube to four cats (DFG, 1957).
Special studies on teratogenicity
Groups of unspecified numbers of mice received daily doses of
7.5, 30 or 100 mg/kg bw orange RN by gavage either from day 0-7 or
day 6-18 of pregnancy. Litter parameters and implantation showed no
adverse effects. No dose-related abnormalities were seen in the
fetuses (Larsson, 1974).
Animal Route mg/kg bw
Mouse Oral 8 000 Gaunt et al., 1967
i.p. > 1 750 approx. Gaunt et al., 1967
Rat Oral >8 000 Gaunt et al., 1967
i.p. 2 000 approx. DFG, 1957
Female i.p. 2 600 Gaunt et al., 1967
Male i.p. 600 Gaunt et al., 1967
i.v. 1 000 DFG, 1957
Groups of 16 male and 16 female rats were fed diets containing
0, 0.5%, 1% and 2% dye for 90 days. No adverse effects were seen in
appearance, behaviour, growth, food consumption, haematological
indices, SGPT and SGOT serum levels except at 2% level, when females
had slightly increased transaminase values, red cell counts and
haemoglobin concentration. Renal function tests and organ weights were
normal. Gross and histopathology showed no difference between the test
groups (Gaunt et al., 1967).
Eleven rats were given 1.0% of the colour in their drinking water
for 216 days and observed for 791 days. Two animals died during the
experiment, one had a sarcoma of the liver (DFG, 1957).
Four groups of three large white pigs of each sex were fed 0
(control), 100, 300 or 900 mg Ponceau 4R/kg bw/day for three months.
One female from the highest dose level died on day 23, but the death
was attributed to an enteric infection. No differences between treated
and control pigs were detected in growth, composition of urine and
serum, organ weights or histopathology. There was a slight reduction
in the number of erythrocytes at week 6 in males for 900 mg/kg/day
(Gaunt et al., 1969).
Groups of 30 mice of each sex (Ash-CS1 strain) were fed on diets
containing 0.01, 0.05, 0.25 and 1.25% Ponceau 4R for 82 weeks with a
group of 60 mice of each sex serving as controls. There was evidence
that the colouring was converted to a yellow metabolite in the
gastrointestinal tract. The feeding of Ponceau 4R had no adverse
effect on mortality, body weight gain, organ weight or the incidence
of tumours. A mild anaemia was present in the first six months of the
study in mice given 0.25 or 1.25% Ponceau 4R. There was an increased
incidence of foamy reticulo-endothelial cells in the liver at the
1.25% level and an increased incidence of glomerulonephrosis at this
and the 0.25% level.
This study shows that the colouring has no carcinogenic potential
in mice when fed at dietary concentrations of up to 1.25%. The no-
untoward-effect level was 0.05% (Gaunt et al., 1974).
Ten rats were given 0.2% of the colour in their diet for 417
days. The total intake was 11 g/animal. Observation extended for 1011
days. One rat died. No tumours were found (DFG, 1957).
In a similar experiment the dye was given to 11 rats as a 1%
solution in the drinking water, the daily dose being 1 g/kg for a
period of 216 days. Total ingestion amounted to 52 g/animal. Animals
were observed for 791 days. One rat developed a sarcoma in the liver,
two others died (DFG, 1957).
Four groups of 10 male and 10 female rats were given diets
containing 0, 0.03%, 0.3%, and 3.0% of the colour for 64 weeks. No
effect was noted on mortality. Females at the highest level had a
lower food consumption throughout the experiment than the controls
with a significant decrease in body weight at 16 and 64 weeks. In
females the relative weights of heart, liver and kidney were
increased. No effects were found on histopathology and as regards
haemoglobin levels (Allmark et al., 1957).
Seventy-five rats were fed the colour at a level of 0.1% of the
diet. No tumours were observed. Similar results were obtained with 10
rats fed at 0.2% of the diet. Feeding extended for life span (DFG,
Thirteen rats were given twice weekly s.c. injections of 0.5 ml
of 1.0% of the colour for 365 days. Observation extended for 857 days.
Five animals died during the experiment. No tumours were found (DFG,
Several long-term studies specifically designed for
carcinogenesis only have been performed in the rat, and another 80
week study in the mouse is now available. Teratology has been examined
in the mouse and a short-term study in a non-rodent species has been
performed. There is little information on the metabolism of this
Level causing no toxicological effect
Mouse: 0.05% (= 500 ppm) in the diet equivalent to 25 mg/kg bw.
Estimate of acceptable daily intake for man
0-0.125 mg/kg bw*
FURTHER WORK OR INFORMATION
Required by June 1978
Metabolic studies in several species preferably including man.
Adequate long-term study in another species and reproduction studies.
Allmark, M. G., Mannell, W. A. & Grice, H. C. (1957) J. Pharm.
Pharmacol., 9, 622
Bar, F. & Griepentrog, F. (1960) Med. u. Ernähr., 1, 99
Deutsche Forschungsgemeinschaft, Farbstoff Kommission (1957)
Gaunt, I. F. et al. (1967) Fd. Cosmet. Toxicol., 5, 187
Gaunt, I. F. et al. (1969) Fd. Cosmet. Toxicol., 7, 443
Larsson, K. S. (1974) Unpublished report submitted by National Food
Administration, Stockholm, to WHO
Lück, H. & Rickerl, E. (1960) Z. Lebensmitt.-Untersuch., 112, 157
Mason, P. L. et al. (1974) Fd. Cosmet. Toxicol. (In press)
Ryan, A. J. & Wright, S. E. (1961) J. Pharm. Pharmacol., 13, 492
Walker, R. (1968) Ph.D. Thesis University of Reading - cited by Gaunt,
I. F. et al. (1969)