PONCEAU 4R
Explanation
This colour was evaluated for acceptable daily intake by the
Joint FAO/WHO Expert Committee on Food Additives in 1964, 1969, 1974,
1978 and 1981 (see Annex I, Refs. 8, 19, 34, 48 and 57). Toxicological
monographs were issued in 1964, 1969, 1974 and 1978 (see Annex I,
Refs. 10, 20, 35 and 49). 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.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Absorption, distribution, excretion and metabolism
Ponceau 4R is reduced in vitro by rat caecal contents (Walker,
1968), but Ryan & Wright (1961) showed that rats excreted 30-45% of an
intravenous dose unchanged in the bile within six hours. The bile was
coloured in rats and mice given intraperitoneally doses of the dye
(Gaunt et al., 1967). The absorption, metabolism and excretion of
orally administered 14C-labelled Ponceau 4R has been studied in male
and female Wistar albino rats, male CD-1 mice and male Dunkin-Hartley
guinea-pigs. Following administration of a single oral dose of either
0.5 mg/kg or 50 mg/kg (20 µCi/kg bw), substantially all of the dose
was excreted in urine and faeces within 72 hours, with the majority
being accounted for in the faeces (90%) and less than 0.003% of the
higher dose eliminated as 14CO2. In all three species studied,
naphthionic acid was the major urinary metabolite, whereas naphthionic
acid, 7-hydroxy-8-amino-naphthalene-1,3-disulfonic acid and unchanged
dye were found in faeces. Pretreating male rats with unlabelled
colouring in the diet (50 mg/kg/day) for 28 days prior to dosing with
14C-labelled colouring (20 µCi/kg) had no effect on the route of
excretion or the time taken to eliminate all of the label. Following a
single dose of 14C-labelled colouring to previously untreated rats,
mice and guinea-pigs or rats given repeated doses of unlabelled
colouring (50 mg/kg/day for 28 days), no marked accumulation of
radioactivity in any tissue was found. Although pregnant rats
eliminated a single oral dose of 14C-labelled colouring at a similar
rate to non-pregnant females, some retention of radioactivity in the
foetuses was found (0.3-0.5%). No significant absorption of Ponceau 4R
was detected using isolated loops of small intestine of the rat,
containing concentrations of colouring of 50, 500 or 5000 ppm (0.005,
0.05 or 0.5%). Some absorption was seen from isolated loops of mouse
intestine at the lowest concentration, and from the guinea-pig at the
two higher concentrations. Less than 0.1% of the administered
radioactivity in the 50 mg/kg dose was recovered in the bile during
one hour and between 0.6% and 1.3% during five hours. Two radioactive
components were present in the urine of all three species. The major
component (70% of the radioactivity in the rat) present in the
methanol extract co-chromatographed with naphthionic acid in three
systems. The other component had a similar chromatographic mobility to
7-hydroxy-8-amino-naphthalene-1,3-disulfonic acid. A third (minor)
radioactive component seen in mouse urine co-chromatographed with
Ponceau 4R. Thin-layer radio-chromatography of faecal extracts from
rats, mice and guinea-pigs given 50 mg 14C-labelled Ponceau 4R/kg
showed that three radioactive compounds were present. The compounds
co-chromatographed with naphthionic acid, 7-hydroxy-8-amino-
naphthalene-1,3-disulfonic acid and unchanged Ponceau 4R. In all three
species approximately 25-35% of the administered dose of colouring
(due correction being made for recovery of radioactivity (80%)) was
excreted unchanged in the faeces during 24 hours (Phillips et al.,
1982).
TOXICOLOGICAL STUDIES
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). The mutagenicity of Ponceau 4R has
been tested in vitro using mutant strains of Bacillus subtilis
with (rec+) and without (rec-) the ability to repair DNA by
recombination mechanisms. No increase in lethatity of bacteria was
observed which indicates that Ponceau 4R does not react with DNA (Kada
et al., 1972). Cultured rat foetal hepatocytes were used to test 11
different food colours for cytotoxicity. Ponceau 4R was rated as
moderately toxic (Sako et al., 1980).
Special studies on reproduction
Ponceau 4R was given in the diet of Wistar rats over three
generations at concentrations to provide 0, 50, 500 or 1250 mg/kg/day.
Animals of both sexes were used in groups of 36 for treated and 60 for
control groups. The parent generation (F0) were given Ponceau 4R from
the time they were weanlings and the succeeding generations were
exposed throughout their lifetimes, including in utero, since
treatment continued during the gestation and lactation periods. The
parental generation was bred twice to provide the F1a and F1b
generations. The former were used to provide the subsequent generation
and animals for a long-term study. The second breeding provided data
covering in utero and post-partum development. The foetuses of the
F1b, F2 and F3 generations were examined for skeletal development
whereas those allowed to litter were monitored over the first 21 days
of life for survival and developmental milestones. All animals were
killed and subjected to a post-mortem examination which, for a
selection of animals from each generation, included weighing of organs
and, for the F3 generation, a histological examination of the
tissues.
During the study, eight females and two males involving all three
generations of adult rats (F0, F1a and F2) died or were killed
because of sickness. The post-mortem examinations of these animals,
the majority of which were females in the later stages of pregnancy,
did not reveal any findings that could be associated with treatment. A
general observation made throughout the study was the pink coloration
of the fur of animals exposed to Ponceau 4R and softer more yellow-
coloured faeces from those given 500 and 1250 mg/kg/day Ponceau 4R. A
yellow coloration was also seen in the caecal contents of the treated
animals and was attributed to the presence of a yellow metabolite.
The clinical observations and records of body weight, food intake
and water intake during the three pre-mating feeding periods did not
show any changes that could be associated with treatment. The various
post-mortem examinations showed a pink colouring of the
gastrointestinal tract at all dose levels. There were enlarged caeca
in animals of the two higher dose levels after prolonged feeding but
not in weanling animals, and lower liver weights in F2 and F3
offspring of the same dose levels. These findings were not considered
to represent an adverse effect. Examination of the uterine contents of
pregnant females of the F0, F1a and F2 generations revealed no
treatment-related differences in the incidence of pre- and post-
implantation losses or of the weight and appearance of the foetuses.
The only finding from the examination of the stained skeletons of
these young indicated a slightly more advanced development in those
from the treated groups. The postnatal development of the offspring
from females allowed to litter, as judged by survival, body weight and
developmental milestones, was not affected by treatment. There were no
differences between the untreated animals and those given 1250 mg
Ponceau 4R/kg/day in the histological examination of the tissues from
the F3 generation. It is concluded that this study has shown a lack
of any adverse effects of Ponceau 4R fed up to 1250 mg/kg/day over
three generations. The reproductive performance of the adults, the
in utero development of the young and the post-partum development of
the pups were normal (Stevenson et al., 1980).
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 NMRI mice received daily doses
of 7.5, 30 or 100 mg/kg bw Ponceau 4R by gavage either from days 0 to
7 or days 6 to 18 of pregnancy. The foetuses were removed on day 18
for detailed examinations. No effect of treatment with the dye could
be observed with regard to number of implantations, frequency of
foetal death and resorptions, gross malformations, skeletal or
internal malformations, or retarded growth as judged by foetal weight
(Larsson, 1975). An embryotoxicity study on Ponceau 4R was carried out
in an unspecified number of SPF Wistar rats. The substance was given
by gavage from days 1 to 20 of the gestation period in doses of 0,
1000, 2000 and 4000 mg/kg bw per day, dissolved in distilled water.
The foetuses were removed on day 21. No effect due to the treatment
with the dye was observed with regard to the number of corpora luteae
(CL), of implantations, of foetuses dead or alive, with regard to
gross malformations, to skeletal and internal malformations, or to the
weight of the foetuses (Meyer & Hansen, 1975). Pregnant Wistar rats
were fed a diet containing 0, 0.01, 0.1 or 1% Ponceau 4R throughout
the gestation period. Observation of the foetuses at term revealed no
deleterious effects due to the treatment with the dye in regard to
embryonic death or intrauterine growth. Gross skeletal and visceral
abnormalities were not generally noted except for a decrease in the
number of ossified phalanges of the hind limb in the 0.01% dye-treated
group. In the postnatal study, survival index, body weight gain,
skeletal development and external differentiation were similar between
the dye-treated groups and the controls. However, the 1% dye-treated
group showed an increased incidence of dilated renal pelvises in the
offspring observed at eight weeks after birth. In order to re-examine
this problem, pregnant rats of the same strain were fed a diet
containing 0, 0.01 or 1% Ponceau 4R throughout the gestation period
and were allowed to deliver their young. Kidneys of the offspring were
examined macroscopically at four days, three weeks, 10 weeks and 22
weeks after birth. Dilated renal pelvises were observed in each group
including the controls, but no significant difference in incidence was
shown between the dye-treated groups and the controls. Histological
examination of the kidneys with enlarged pelvises showed no remarkable
pathological changes. No other kidney anomalies were found in the
dye-treated groups. In conclusion, Ponceau 4R is considered to show no
adverse effects on prenatal and postnatal development in rats (Kihara
et al., 1977).
Acute toxicity
Animal Route LD50 Reference
(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
Short-term studies
Rat
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 the 2% level, when
females had slightly increased transaminase values, red-cell counts
and haemoglobin concentration. Renal function tests and organ weights
were normal. Gross pathology and histopathology showed no difference
between the test groups (Gaunt et al., 1967). Eleven rats were given
1% 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).
Pig
Four groups of three large white pigs of each sex were fed 0
(control), 100, 300 or 900 mg Ponceau 4R/kg bw per 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 given
900 mg/kg/day (Gaunt et al., 1969).
Long-term studies
Mouse
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 reticuloendothelial 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 up to 1.25%. The
no-untoward-effect level was 0.05% (Mason et al., 1974).
Rat
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% 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 rumours were observed. Similar results were obtained with 10
rats fed at 0.2% of the diet. Feeding extended for life span (DFG,
1957). Thirteen rats were given twice weekly s.c. injections of 0.5 ml
of 1% of the colour for 365 days. Observation extended for 857 days.
Five animals died during the experiment. No tumours were found (DFG,
1957).
Groups of 66 male and female Wistar rats were given diets to
provide 50, 500 or 1250 mg Ponceau 4R/kg/day for nine weeks and 114
males and females were given basal diet to serve as controls. Each
female was paired with a male of the same dose group for 13 days and
treatment continued during pregnancy and pup-rearing periods. Young
were selected on the basis of one male and one female per litter to
give group sizes of 54 and 96 of each sex for the treated and control
groups respectively. The selection was made so that the treatment of
the young was the same as their parents and it continued until only
approximately 20% of the animals survived. This resulted in a maximum
treatment duration of 114 weeks for the males and 118 weeks for the
females. The fur of all treated animals was coloured pink due to
contamination with Ponceau 4R, but, apart from this finding, none of
the observations on the parental animals, including those of fertility
and pup rearing, could be related to treatment.
The young exposed to 50, 500 or 1250 mg Ponceau 4R/kg/day for
their life span showed no difference of survival. There was a slightly
lower weight gain in males and females given 1250 mg/kg that was not
due to a lower food intake. The animals given 500 or 1250 mg/kg/day
had soft and unformed faeces thought to be associated with an increase
in the weight of the wall of the caecum and its contents and
elimination of water with the faeces. This water loss was in turn
considered to be responsible for an increase in water consumption in
the same animals. There were no changes that could be related to
treatment in the haematological examinations at 3, 6, 12, 18 and 24
months and at the end of the study, and none that could be considered
to represent an adverse effect of treatment in the renal concentration
tests or measurement of cell excretion and urinary pH at 3, 6, 12, 18
and 24 months. At 18 and 24 months, the incidence of females with high
concentrations of protein in the urine was greater in the group given
1250 mg/kg/day than in the controls.
Kidney weight was elevated, not dose related, in the males given
the lowest (+123%) and the highest (+128%) levels of treatment with no
comparative effect in the females and no histological findings that
were associated with this increase in weight. Testes weight was
elevated at the two highest dose levels (+114 and +133% respectively).
With the exception of the higher caecum and kidney weights, the
remaining organ weights were not influenced by Ponceau 4R. The
histopathology did not reveal any differences between the control and
treated animals that could be related to treatment and neither did the
analysis of serum and plasma of animals killed at the end of the
study. The types and incidence of tumours were those expected for this
strain of rat and there was no evidence of a treatment-related
increase in the number of animals with each finding or in the total
number of animals with benign and malignant tumours. It is concluded
that Ponceau 4R was not carcinogenic to rats fed 1250 mg/kg/day for
the bulk of their life span. The no-untoward-effect level was
500 mg/kg/day (Stevenson et al., 1981).
OBSERVATIONS IN MAN
In a series of 51 patients showing signs of allergy, the authors
showed that 16% reacted to an oral dose of Ponceau 4R. This reaction
rate was no more than was seen with other synthetic colours and was
less than with aspirin or a natural colour (Annatto extract). No
evidence was presented to show if the colour was the sensitizer or if
it was a cross-reaction with some other material (Mikkelson et al.,
1978). Patch tests were carried out on 17 different colouring matters
including Ponceau 4R using 50 patients who had been given a
presumptive diagnosis of possible allergic contact dermatitis to
coloured dyes. None of the patients tested demonstrated sensitization
to Ponceau 4R (Rapaport, 1980).
Comments
Metabolism studies indicate that Ponceau 4R is rapidly absorbed
from the gastrointestinal tract and undergoes extensive metabolism
with the formation of naphthionic acid and 7-hydroxy-8-amino-
naphthalene-1,3-disulfonic acid. Following a single dose of
14C-labelled colouring to previously untreated rats, mice and guinea-
pigs or rats given repeated doses of unlabelled colouring for 28 days,
no marked accumulation of radioactivity in any tissue was found.
Pregnant rats eliminated a single oral dose of 14C-Ponceau 4R at a
similar rate to non-pregnant females. Following administration of a
single oral dose to male and female rats, mice and guinea-pigs,
substantially all of the dose was excreted in urine and faeces within
72 hours, with the majority being accounted for in the faeces (90%). A
three-generation reproduction study in the rat indicates that the
clinical observations, body weight, food and water intake during the
feeding periods did not show any changes that could be associated with
treatment. Examination of the uterine contents of pregnant females and
the F0, F1a and F2 generations revealed no treatment-related
differences in the incidence of pre- and post-implantation losses or
of the weight and appearance of the foetuses. The reproductive
performance of the adults, the in utero development of the young and
the post-partum development of the pups were normal. It is concluded
that this study has shown an absence of any adverse effects of Ponceau
4R in rats fed up to 1250 mg/kg bw per day over three generations.
Teratogenicity studies did not reveal any compound-related adverse
effects. No mutagenic or cytotoxic effect was found when the colour
was tested in vitro experiments. An adequate long-term study has
been carried out in the rat. There were no changes that could be
related to treatment. The histopathology did not reveal any
differences between the control and treated animals and neither did
the analysis of serum and plasma of animals killed at the end of the
study.
EVALUATION
Level causing no toxicological effect
Mouse: 0.25% (2500 ppm) in the diet, equivalent to 375 mg/kg bw.
Estimate of acceptable daily intake for man
0-4.0 mg/kg bw.
REFERENCES
Allmark, M. G., Mannell, W. A. & Grice, H. C. (1957) Chronic toxicity
studies of food colours. III - Observations on the toxicity of
malachite green, new coccine and nigrosine in rats, J. Pharm.
Pharmacol., 9, 622-628
Bär, F. & Griepentrog, F. (1960) Die Allergenwirkung von Fremden
Stoffen in der Lebensmitteln, Med. u. Ernähr., 1, 99-104
Deutsche Forschungsgemeinschaft (1957) Farbstoff Kommission,
Mitteilung 6, 2, Auflage Toxikologische Daten von Farbstoffen und
ihre zullassung fur Lebensmittel in verschiedenen Landern, Franz
Steiner Verlag GmbH, Wiesbaden, p. 38
Gaunt, I. F. et al. (1967) Acute (mouse and rat) and short-term (rat)
toxicity studies on Ponceau 4R, Food Cosmet. Toxicol., 5,
187-194
Gaunt, I. F. et al. (1969) Short-term toxicity on Ponceau 4R in the
pig, Food Cosmet. Toxicol., 7, 443-449
Kada, T., Tutikawa, K. & Sadate, Y. (1972) In vitro and host-
mediated "rec-assay" procedures for screening chemical mutagens
and phloxine, a mutagenic red dye detected, Mutation Res.,
16, 165
Kihara, T., Yasuda, Y. & Tanimura, T. (1977) Effects on pre- and post-
natal offspring of pregnant rats fed Food Red No. 102, Abstract,
Teratology, 111-112 (December)
Larsson, K. S. (1975) A teratologic study with the dyes Amaranth and
Ponceau 4R in mice, Toxicology, 4, 75-82
Lück, H. & Rickerl, E. (1960) Food additives and mutative effects. VI
Report, Z. Lebensmitt.-Untersuch., 112, 157-174
Mason, P. L. et al. (1974) Long-term toxicity study of Ponceau 4R in
mice. Unpublished report No. 1/1974, submitted to WHO by the
British Industrial Biological Research Association
Meyer, O. & Hansen, E. V. (1975) A study of the embryotoxicity of the
food colour Ponceau 4R in rats, Toxicology, 5, 201-207
Mikkelson, H., Larsen, J. C. & Tarding, F. (1978) Hypersensitivity
reactions to food colours with special reference to the natural
colour Annatto extract (butter colour), Archs Toxicol., Suppl.
1, 141
Phillips, J. C., Bex, C. S. & Gaunt, I. F. (1982) The metabolic
disposition of 14C-labelled Ponceau 4R in the rat, mouse and
guinea-pig, Fd Chem. Toxicol., 20, 499-505
Rapaport, M. J. (1980) Patch testing of colour additives, Contact
Dermatitis, 6, 231
Ryan, A. J. & Wright, S. E. (1961) The excretion of some azo dyes in
rat bile, J. Pharm. Pharmacol., 13, 492-495
Sako, F. et al. (1980) Cytotoxicity of food dyes on cultured fetal rat
hepatocytes, Toxicol. Appl. Pharmacol., 54, 285-292
Stevenson, B. I. et al. (1980) Multigeneration toxicity study in rats
with Ponceau 4R, Vol. I. Unpublished report No. 1/205/80, from
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to WHO by the UK Colours Steering Group
Stevenson, B. I. et al. (1981) Long-term study in rats with Ponceau 4R
using animals exposed in utero, Vol. I. Unpublished report No.
293/2/81, from the British Industrial Biological Research
Association, submitted to WHO by the UK Colours Steering Group
Walker, R. (1968) Ph.D. Thesis, University of Reading - cited by Gaunt
et al. (1969)
See Also:
Toxicological Abbreviations
Ponceau 4R (FAO Nutrition Meetings Report Series 46a)
Ponceau 4R (WHO Food Additives Series 6)
Ponceau 4R (WHO Food Additives Series 13)
PONCEAU 4R (JECFA Evaluation)