ERYTHROSINE
EXPLANATION
Erythrosine was evaluated for an acceptable daily intake by the
Joint FAO/WHO Expert Committee on Food Additives at its eighth,
thirteenth, eighteenth, and twenty-eighth meetings (Annex 1,
references 8, 19, 35, and 66). Toxicological monographs were published
after the thirteenth, eighteenth, and twenty-eighth meetings (Annex 1,
references 20, 36, and 62). At its eighteenth meeting the Committee
allocated an ADI of 0-2.5 mg/kg b.w.; this was reduced at the
twenty-eighth meeting to 0-1.25 mg/kg b.w. and made temporary
following observtions that erythrosine produced effects on thyroid
function in short-term studies in rats and that, in long-term studies,
male rats receiving 4% erythrosine in the diet developed thyroid
tumours (begnin follicular adenomas). The Committee requested further
information on the histopathology (including diffuse hyperplasia) of
all thyroid glands from the long-term studies in rats, the mechanism
of the effects of erythrosine on the thyroid, and the existence of a
threshold and reversibility of these effects. It was also considered
desirable to have further information on the pharmacokinetics of
erythrosine and its effect on thyroid function in human subjects.
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
The metabolic behaviour and excretory pattern of erythrosine have
been studied in adult rats. When the colour was given by stomach tube
to male rats at levels as high as 150 mg/rat, recovery in the excreta
after 5 days was 102%. After i.v. administration of 3 mg erythrosine
per kg b.w., an average of 55% of the administered quantity was found
in the bile and 1.3% was recovered in the urine. No glucuronic acid
conjugation was found (Daniel, 1962).
In studying the possibility that iodide may be liberated from
erythrosine and disturb thyroid function in the rat, erythrosine was
found to be metabolically stable; 100% of the amount ingested was
excreted, with its iodine content intact, after administration of
500 mg/kg erythrosine (Webb et al., 1962).
The urinary and faecal excretion of 14C-labelled and of
125I-labelled erythrosine were studied in rats of both sexes either
without pre-treatment or following dosing with unlabelled erythrosine
at dietary levels of 0.5 or 4.0% for 7 days. The distribution of the
compound in tissues and body fluids was also studied.
The radioactivity from both radiolabels was excreted
predominantly in the faeces, mainly within 48 hours; less than 1% of
the dose was excreted in urine. Blood and plasma radioactivity reached
maximum levels by 1 hour, while levels in the liver and kidneys peaked
after 4-12 hours. The activity in blood and tissues was very low,
suggesting that erythrosine is not extensively absorbed from the
gastrointestinal tract. Of the tissues examined (liver, kidney,
thyroid, brain, and pituitary), the highest levels of radioactivity
were found in the liver (maximally 0.145% of the dose of 14C; 0.188%
of the dose of 125I). Thyroid residues of 14C were at trace or
non-detectable levels, while levels of 125I were detectable but low
(maximally approximately 0.01% of the dose), indicating that neither
erythrosine nor its ring-containing metabolites accumulated in the
thyroid. The magnitude of the 125I levels in the thyroid was so low
that it was not possible to conclude whether the activity resulted
from 125I-iodide in the dose or from 125I-iodide formed by a smell
degree of metabolic deiodination of erythrosine. No 14C or 125I was
detectable in the brain or pituitary. Smell amounts of metabolites,
believed to be isomeric diiodo- and triiodofluoresceins, were detected
in urine, faeces, plasma, and tissue extracts from the liver and kidney
(Obrist et al., 1986).
Protein-bound and total blood iodine levels were elevated in rats
given erythrosine by stomach tubs twice weekly in a chronic study.
However, in experiments with rats and gerbils, it was concluded that
the elevated protein-bound iodine (PBI) was due to interference by
erythrosine in PBI determinations (Bowie et al., 1966; U.S. FDA,
1969).
Large doses of erythrosine labelled with 131I given orally to
rats inhibited uptake of 131I by the thyroid of treated animals.
Daily doses of over 1 mg erythrosine were necessary for this effect to
occur (Harignan et al., 1965).
Studies were performed to investigate the effects of erythrosine
on the metabolism of 125I-labelled thyroxine (T4) and
triiodothyronine (T3) by rat liver homogenates. The rats were given
i.p. daily doses of 2.5 - 250 mg erythrosine/kg b.w. in vivo prior
to preparation of the liver homogenates. Erythrosine caused a
dose-dependent inhibition of the deiodination of T4 and of the
generation of T3. At dose levels of > 10 mg/kg b.w., the
proportionate reduction in the deiodination of 125I-T4 exceeded
the reduction in the generation of 125I-T3, indicating that
pathways of T4 metabolism other than those leading to T3 formation
were also inhibited. The authors concluded that erythrosine may
inhibit the 5-monodeiodinat ion of T4 to reverse T3 (rT3) since
erythrosine also inhibited the 5-monodeiodination of T3 to
3,3'-diiodothyronine. Fluorescein did not exhibit similar effects. It
was considered unlikely that erythrosine would produce similar effects
in man in the doses normally ingested. This conclusion was supported
by studies in human volunteers given oral doses of erythrosine of up
to 25 mg daily for 7 days when no changes in serum T4, T3, or
rT3 concentrations were detected (Ruiz & Ingbar, 1982;
Garber et al., 1981).
Six groups of 15 male Sprague-Dawley rats received 0, 0.25, 0.50,
1.0, 2.0, or 4.0% erythrosine in the diet for a period of 7 months.
After 6 months each group was divided into three sub-groups of 5 rats;
during the next month one sub-group received 15 µg T3/kg b.w./day by
s.c. injection, a second sub-group received the saline vehicle, and
the third sub-group continued with no injections. Blood samples
(orbital puncture) were obtained just before the start of the study
and at monthly intervals thereafter and serum thyroid hormone levels
were determined. At termination, pituitary glands and liver segments
were excised and in vitro studies of T4 metabolism were performed
on these tissues.
Serum thyrotropin (TSH) levels were variable and, although mean
serum TSH values were higher in the 4% erythrosine group than in the
control or 0.5% groups over the first 6 months of the study, the
differences were not statistically-significant. Serum TSH
concentrations in sub-groups which received injections of T3 during
the final month were below detection limits (15 µU/ml). Serum T4
concentrations were elevated relative to base-line and control values
in animals receiving 4% erythrosine during the 6 months of the study,
whereas the values for the control and 0.5% erythrosine groups did not
differ significantly from base-line values or from each other. Serum
T4 concentrations were immeasurably low in all animals that had
received injections of T3 during the seventh month of the study.
Serum T3 concentrations in all 3 groups decreased significanlty with
time; additionally, 4% erythrosine in the diet decreased the T3
concentration and the values were significantly lower than controls at
1, 2, 4, and 5 months. The effects of 0.5% erythrosine on T3 levels
were unclear, significant depressions relative to controls only being
observed at the 1- and 2-month time periods and not subsequently. In
animals receiving 4% erythrosine, serum rT3 concentrations increased
approximately 7-fold and remained elevated throughout the 6 months of
the study. In both control and 0.5% groups, serum rT3 concentrations
did not differ significantly from base-line values or from each other.
Serum rT3 was undetectable in animals receiving injections of T3
during the final month of the study.
In vitro metabolism of 125I-labelled T4 was greatly altered
in liver homogenates from rats fed 4.0% erythrosine in the diet, with
degradation of T4 decreasing to approximately 40% of values in
homogenates of control livers. There was an associated decrease of
about 75% in the generation of 125I-iodide and an approximately 80%
decrease in the generation of 125I-T3 from 125I-T4. Percentage
degradation of T4 and generation of iodide and T3 in liver
homogenates from rats fed 0.5% erythrosine were similar to controls.
In vitro metabolism of T4 was studied in pituitary glands
from the control, 1, 2, and 4% dose groups. Overall, 125I-T4
degradation and generation of 125I-iodide appeared higher in the two
higher erythrosine dose groups, than in controls, but none of the
differences were statistically significant.
The results were interpreted as indicating that the primary
effect of high doses of erythrosine on thyroid hormones is inhibition
of type I 5'-monodeiodination of T4 to T3. As a consequence, TSH
secretory mechanisms were activated in the pituitary. The increases in
serum rT3 levels were considered to arise from both increased
availability of the T4 precursor and inhibition of metabolism of
rT3 by 5'-monodeiodination (Ingbar et al., 1984a).
In identical studies on female rats to those outlined above,
similar results were obtained in that erythrosine at dietary
concentrations of 4% caused an increase in serum T4, rT3, and TSH
concentrations, a decrease in hepatic deiodination of T4 to T3, and
an increase in deiodination in the pituitary. Hepatic generation of
T3 from T4 was also diminished following dietary administration of
0.5% erythrosine, but to a lesser degree and no changes in serum
thyroid-related hormones could be detected. No alteration in the
metabolism of T4 was observed in liver homogenates from rats
receiving 0.25% dietary erythrosine (Ingbar, 1985).
In both the above studies, ultrastructural examination of
thyroids of rats receiving 4% erythrosine in the diet for 6 months
revealed enhanced synthetic and secretory activity consistent with
prolonged hyperstimulation by TSH. Less marked changes were seen at
the 0.5% dose level and the changes generally were less in females
than males similarly treated (see Special studies on thyroid
morphology).
Groups of 13 adult male Sprague-Dawley rats were fed 0, 0.5, 1,0,
or 4.0% erythrosine in the diet, 100 mg sodium iodide/kg b.w./day, or
1000 mg fluorescein/kg b.w./day for three weeks. The rats were then
subjected to an in vivo TSH-releasing hormone (TRH) provocative test
(100 ng/100 g b.w.). Of all the treatments, only 4% dietary
erythrosine produced an exaggerated response to TRH; ten minutes after
TRH injection, the serum TSH levels were 80% greater than controls
(p < 0.01). Erythrosine also produced a dose-dependent increase in
total serum T4 levels, significant (p < 0.01) at the 1% and 4% dose
levels and, at the 4% level, there was also a significant increase in
serum T3 levels and a significant decrease in T3 resin uptakes.
The free T4 indices were significantly elevated after treatment with
1% or 4% dietary erythrosine, but the free T3 indices were not. The
data were taken to indicate that feeding with 4% erythrosine disrupts
the normal negative feedback regulatory mechanism of the pituitary-
thyroid axis and the TRH hyper-responsiveness in the presence of elevated
serum T4 and T3 levels suggested that the defect did not arise
from a conventional antithyroid mechanism. The data also suggested that
the effect of erythrosine on TSH release was due to intact dye or an
iodinated metabolite rather than to the fluorescein nucleus (Witorsch
et al., 1984).
In man, oral administration of 16 mg of erythrosine daily for 10
days resulted in an increase from 6 to 11 µg of protein-bound iodine
per 100 ml serum after 15 to 20 days, followed by a sharp decline in
iodine levels in the next 10 days, with a gradual return to the
initial value in three months (Anderson et al., 1964).
Erythrosine could be an adventitious source of iodide (Vought
et al., 1972).
No biologically-significant increases in plasma inorganic iodine
or in urinary iodine were found in 6 patients (aged 25-68 years, sex
not reported) after oral exposure to 1.9 µmole (1.68 mg/day) of
erythrosine for 10 days. In other assays of thyroid function, thyroid
radioiodine uptake, levels of thyroxine, and PBI in plasma remained
unchanged (Bernstein et al., 1975).
When cherries coloured with erythrosine are stored in plain cans,
fluorescein is readily formed by interaction of the tin-iron couple
that is present. This does not occur in lacquered cans. The production
of fluorescein from erythrosine occurs in the presence of metallic
iron and/or tin and free organic acid as a result of electrochemical
reduction in the can (Dickinson & Raven, 1962).
It was found that 200-400 mg/l erythrosine inhibited the action
of pepsin, but had no effect on lipase activity (Diemair & Hauser,
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 (Waliszewski, 1952).
Erythrosine was administered twice weekly to rats in doses of 5,
10, 15, or 50 mg per rat weighing 200-250 g for 6 months. Haemoglobin
and red cell counts were reduced at 3 months. The cholesterol levels
of males were depressed. Excretion of the dye was mainly in unchanged
form in the faeces (Bowie et al., 1966).
Toxicological studies
Special studies on carcinogenicity
See under long-term studies.
Special studies on mutagenicity
Erythrosine was tested for mutagenic activity and showed a very
slight but statistically-significant mutagenic effect on Escherichia
coli at a concentration of 0.5 g/100 ml. It was found that the
xanthene molecule itself was the causative factor and that the
substituent groups only modify the effect (Luck et al., 1963;
Luck & Rickerl, 1960).
No mutagenic activity was observed using E. coli WP2 UVrA as
the indicator organism (Haveland-Smith & Combes, 1980).
A lack of mutagenic activity of erythrosine in Salmonella
typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 was
observed when tested in the Ames test at concentrations ranging from 1
to 10,000 µg/plate with or without metabolic activation (Auletta
et al., 1977; Bonin & Baker, 1980; Brown et al., 1975).
Similar negative results in the Ames Test were obtained in
several other recent studies (Tarján & Kurti, 1982; Ishidate et al.,
1984; Jaganath & Myth, 1984a; Muzzall & Cook, 1979).
Erythrosine was inactive in the host-mediated rec-assay
(Kada et al., 1972) and in a mouse host-mediated assay using
Salmonella typhimurium strains TA98, TA100, and TA1537 (Tarján &
Kurti, 1982).
Erythrosine was found to be non-mutagenic in a mouse lymphoma
L5178Y TK+/- forward mutation assay (Cifone & Myhr, 1984) and did not
induce cell transformation in rat embryo cells in vitro or in vivo
(Price et al., 1978).
Erythrosine was inactive in DNA-repair, fluctuation, and
treat-and-plate assays (Haveland-Smith et al., 1981) and in mitotic
gene conversion assays using yeast strain B234 (Sankaranarayanan &
Murthy, 1979) and strain D5 (Jaganath & Myhr, 1984b; Matula & Downie,
1984). Reported positive results in a mitotic gene conversion assay
using yeast strain D7 (Matula & Downie, 1984) and in a reverse
mutation assay in yeast XV185-14C have been called into question
(Brusick, 1984).
Erythrosine was inactive in the mouse micronucleus assay (Tarján
& Kurti, 1982; Ivett & Myhr, 1984). An in vitro chromosome
aberration assay in hamster cells indicated an effect, but this may
have resulted from osmotic effects of the high concentration (5 mg/ml)
of erythrosine at which the observations were noted (Ishidate
et al., 1984).
Special studies on reproduction
Rats
Four groups of Charles River CD rats (23-25 males and
females/group) received erythrosine in the diet at dose levels of 0,
0.25, 1.0, or 4.0% for 3 consecutive generations. The F0 parental
rats received their respective diets for 69 days prior to mating. The
study showed that during the gestation period slight to moderate
reductions in mean maternal body-weight gain were noted in females of
all generations at the 1.0 and 4.0% dose levels. Slight to moderate
reductions in mean pup body weights were recorded at the 4.0% level on
lactation days 0, 4, 14, and 21 in all generations. These reductions
were statistically-significant only on lactation day 21. There were no
consistent compound-related effects on the reproductive performance of
males and females and pup survival at any dose level in any generation
(Albridge et al., 1981).
Groups of 18-22 pairs (males and females, weighing 200-220 g) of
adult Sprague-Dawley rats were fed diets containing erythrosine at
levels of 0, 0.25, 0.5, or 1.0% for 2 weeks before mating and during
the mating period. The diets were continued for the females throughout
gestation and lactation and were provided continuously to their
offspring until they reached 90-100 days. Animals in the positive
control group did not receive erythrosine in the diet, but offspring
were injected daily with 50 mg/kg of hydroxyurea on post-natal days
2-10. Two years later, a second experiment, a replication of the first
one with the same dose groups and number of animals per group was
performed. In both experiments, parental animals were evaluated for
weight and food consumption and females for reproductive success. The
offspring were assessed for behavioural toxicity plus weight, food
consumption, physical development, and brain weight.
Erythrosine produced no reductions in parental or offspring
weight or food consumption. Erythrosine significantly increased
pre-weaning offspring mortality at the 0.5 and 1.0% dose levels in the
first experiment, but not in the second. Mean litter size was not
adversely affected by erythrosine in either experiment. Behaviourally,
erythrosine produced no dose-dependent effects that replicated across
the two experiments. The authors concluded that these studies provided
no evidence that erythrosine, via dietary exposure at levels as high
as 1.0%, is psychotoxic to developing rats (Vorhees et al., 1983).
Special studies on thyroid morphology
Thyroid glands from the Primate Research Institute 27-week
toxicity study (see Couch et al., 1983 study under Long-term
studies) were subjected to ultra-structural examination by electron
microscopy. Rats fed erythrosine were reported as displaying
hypertrophy of follicular cells with increased development of
synthetic and secretory organelles (rough endoplasmic reticulum, Golgi
apparatuses, and long microvilli). These changes were interpreted as
representing mild to moderate stimulation of follicular cells
consistent with elevated serum T4 levels. Lysosomal bodies in rats
receiving erythrosine were described as being larger, more irregular,
and electron dense than controls and appeared to be closely associated
or fused with the limiting membrane of colloid droplets, a process
involved in secretion of thyroid hormones. The degree of thyroid
stimulation and the accumulation of colloid droplets and lysosomes in
follicular cells were stated to be greater in male than in female rats
fed commercial erythrosine. Ultrastructural indications of long-term
thyroid stimulation appeared greater in rats fed commercial
erythrosine than in rats fed purified colour with supplemental iodide
(Capen, sine data a).
Thyroids from rats fed 0, 0.25, 0.5, or 4,0% erythrosine in a
7-month study of thyroid function (see Ingbar et al., 1984 study
under Biochemical aspects) were subjected to examination by electron
microscopy. Thyroid follicular cells from rats fed erythrosine were
reported as displaying ultra-structural features of a dose-dependent
stimulation of synthetic and secretory activity, most marked in rats
fed 4% erythrosine and indicated by hypertrophy of follicular cells
with increased development of secretory organelles. These features
were considered to be generally consistent with a response to
long-standing TSH stimulation. The ultrastructural changes were
reversible by administration of T3 during the last month. A
dose-dependent accumulation of numerous lysosome-like bodies observed
in follicular cells of treated rats were considered not to be an
expected response to TSH stimulation alone. Similar, but less marked
changes, in follicular cell stimulation and accumulation of
lysosome-like bodies were stated to be present in rats fed erythrosine
at dietary levels of 0.25 and 0.5% (Capen, sine data b).
Acute toxicity
LD50
Animal Route (mg/kg b.w.) Reference
Mouse Oral 6800 Butterworth et al., 1976a
i.p. 360 Butterworth et al., 1976a
i.v. 370 Waliszewski, 1952
Rat Oral 1895 Lu & Lavallee, 1964
7100 Butterworth et al., 1976a
1840 Hansen et al., 1973a
i.p. 300 Emerson & Anderson, 1934
350 Butterworth et al., 1976a
Gerbil Oral 1930 U.S. FDA, 1969
Rabbit i.v. 200 Emerson & Anderson, 1934
A group of 5 young rats was given s.c. injections twice daily of
250 mg erythrosine/kg b.w. in aqueous solution for 3 days. The rats
were killed on the fourth day. No oestrogenic activity (based on the
observation that uterine weights were normal) was detected (Graham &
Allmark, 1959).
In experiments with guinea-pigs, it was found that erythrosine
had no sensitization activity (Bär & Griepentrog, 1960).
Short-term studies
Rats
In a 90-day study on 5 groups of 15 male and 15 female rats,
erythrosine was given at levels of 0, 0.25, 0.5, 1, or 2% of the diet.
No adverse effects were noted as regards body weight, food intake,
haematology, or blood and urine analyses which could be related to
administration of the test substance. Organ weights were normal except
that absolute and relative caecal weights were higher at all levels
tested. Caecal enlargement was dose-related, but histology was normal.
Absolute and relative thyroid weights were increased at the 2% level.
Histopathology showed no abnormalities except pigment deposition in
renal tubules in females at the 2% level and in males at all levels in
a dose-related manner. The pigment was identified as protein-bound
erythrosine. In addition, total PHI and protein-bound erythrosine in
serum were raised at all levels in a dose-related manner, and non
protein-bound iodine increased with dose levels. However, T4 iodine
remained unchanged and 131I uptake was reduced (Hansen et al.,
1973b).
Five groups of Carworth farm E strain SPF rats (15 males and 15
females/group) received 0, 0.25, 0.5, 1.0, or 2.01 erythrosine in the
diet for 90 days. There were no effects attributable to treatment on
the rate of body-weight gain or food intake or on the results of
haematological examinations, serum analyses, or renal function tests.
Thyroid weight relative to body weight was slightly increased in rats
receiving 1.0 and 2.0% erythrosine. Thyroid activity was not impaired
at any dietary level of erythrosine. This was indicated by the normal
histopathology of the organ, the lack of effect on serum T4 levels,
and the normal rates of oxygen consumption in the treated animals
(Butterworth et al., 1976a).
Groups of Sprague-Dawley female rats (12-20 animals per group)
were exposed to erythrosine in the diet at dose levels of 0 or 0.2%
for either 6 or 12 months. During the last 12 weeks of the
experimental period, a slight decrease of body-weight gain was
observed in rats exposed for 12 months. Other parameters such as food
consumption, haematology, clinical chemistry, urinalysis, and organ
weights were comparable among treated and control rats in both the
6-and 12-month groups. Sporadic pathological changes were observed in
treated and control rats (Sekigawa et al., 1978).
Gerbils
Three groups of gerbils (15 males and 15 females/group) received
erythrosine in the diet at dose levels of 200, 750, or 900 mg/kg for
19 months (those animals in the 900 mg/kg dosage group received
1200 mg/kg for the first 3 months). The control group consisted of 30
males and 30 females. Body-weight decreases were seen in male gerbils
at all feeding levels. However, this weight loss was observed in
females only at the 900 mg/kg level. Elevated PBIs, due to
interference by erythrosine with PBI determinations, were seen. No
other haematological differences were seen. No adverse gross pathology
was noted. Histopathology was not performed (U.S. FDA, 1969).
Dogs
Two-year feeding studies were conducted with groups of 3 male and
3 female beagle dogs at levels of 0, 0.5, 1.0, or 2.01 erythrosine in
the diet. All dogs survived the study. No gross or microscopic
pathological changes related to colour administration were observed
(Hansen et al., 1973b).
Pigs
Four groups of large white strain pigs (3 males and 3 females/
group weighing approximately 20 kg each) were fed erythrosine
in their diets at dose levels of 0, 167, 500, or 1500 mg/kg b.w./day
for 14 weeks. The treated pigs exhibited decreased levels of serum
T4 when compared with controls. There were dose-related increases in
the serum levels of PBI, non-protein-bound iodine, and protein-bound
erythrosine in animals of all trated groups. A dose- related increase
in thyroid weight was noted, although the differences were
statistically-significant only in female pigs at the higher-dose
levels (500 and 1500 mg/kg b.w./day) when compared with the controls.
None of the treated pigs revealed pathological changes of the thyroid
(Butterworth et al., 1976b).
Long-term studies
Mice
A total of 122 male and female mice aged 50-100 days produced by
mixed breeding from five different strains were given a diet
containing 1 mg of the colour per animal per day. A negative control
group consisting of 168 mice and two positive control groups, which
were given alpha-aminotoluene and dimethylaminoazobenzene, were also
included. A number of the animals were sacrificed after an observation
period of 500 days and the remaining mice were killed after 700 days.
The formation of liver tumours was noted in animals in the positive
control groups after approximately 200 days. The incidence of tumours
in mice receiving the colour was not significantly greater than in the
negative controls (Waterman & Lignac, 1958).
Chronic feeding studies were conducted with mice. Seventy mice
were fed erythrosine at 1 or 2%. Because of the small number of
animals surviving the experiment and the small number of tumours
found, no effect on tumour formation could be attributed to the colour
(U.S. FDA, 1969).
Five groups of Charles River CD-1 mice (60 males and 60
females/group) were exposed to erythrosine in the diet at dose levels
of 0 (2 control groups were used), 0.3, 1.0, or 3.0% for 24 months.
The respective average consumption of erythrosine was 0, 424, 1474, or
4759 mg/kg b.w./day for males and 0, 507, 1834, or 5779 mg/kg b.w./day
for females. With the exception of significantly-decreased body
weights (throughout the entire study) of males and females at the 3.0%
dose level, other investigated parameters (mortality, food intake,
haematology, gross pathology, and histopathology) were not adversely
affected by erythrosine treatment at any dose level (Richter et al.,
1981).
Two groups of 7-week old ICR mice weighing 27-38 g (50 males and
50 females/group) were fed diets containing erythrosine at dose levels
of 1.25 or 2.5% for 18 months. The mice received erythrosine in cube
diet for the first 20 weeks, and thereafter the erythrosine was mixed
with the basic powder diet. All animals in the experimental groups
were fed the basic diet free of erythrosine for an additional 6
months, after which they were sacrificed and autopsied. The control
group consisted of 45 males and 45 females. Mortality was greater
among animals exposed to erythrosine than among the controls
(approximately 61% of the animals died in the 2.5% group, 59% in the
1.25% group, and 36% in the control group). Body-weight gains were not
adversely affected by erythrosine ingestion. Animals in both
experimental groups exhibited a high incidence of lymphocytic
leukemia, and sporadic cases of pulmonary adenomas were observed. The
frequency of both lesions was in the range spontaneously-occurring in
this strain of mice. The results indicate that erythrosine was not
carcinogenic to ICR mice under the experimental conditions utilized
(Yoshii & Isaka, 1984).
Rats
Groups of 24 weanling rats, evenly divided by sex, were fed
erythrosine at 0, 0.5, 1.0, 2.0, or 5.0% for 2 years. Slight growth
depression was observed in the animals at the 5% level. Those animals
fed more than 0.5% erythrosine had distended caeca, but
microscopically the distended caeca showed normal histology. The
statistical evaluation of the rat study revealed no significant
changes in organ weights at the highest level. There was some
diarrhoea at the 5% level. There were no differences in survival among
the test groups (U.S. FDA, 1969).
Erythrosine was fed at a level of 4% of the diet to 5 male and
female rats for periods up to 18 months. Gross staining was observed
in the glandular stomach and small intestine, and granular deposits
were observed in the stomach, small intestine, and colon. Hepatic
cirrhosis was noted in 1 out of 4 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 12 male and 12 female weanling Osborne-Mendel rats were
fed 0, 0.5, 1.0, 2.0, or 5.0% erythrosine in their diet for two years.
Growth depress ion was observed in rats given 5% erythrosine. The
relative spleen weight was depressed in all male test groups and in
females at the 5% level. Slight caecal enlargement was noted at 1%,
which increased with dose, but the histology of the enlarged caeca was
normal. No other gross or hispathological findings related to colour
administration were noted (Hansen et al., 1973b).
Groups of 2.5 male and 25 female 100-day old rats and groups of
50 male and 50 female control rats were fed 0, 0.5, 1.0, 2.0, or 4.0%
erythrosine in their diets for 86 weeks. Other groups of 25 male and
25 female rats aged 100 days were intubated twice a week for 85 weeks
with 0, 100, 235, 750, or 1500 mg erythrosine/kg b.w. After this
treatment, the animals were kept on normal diets for the remainder of
the two years of the study. Body-weight decreases were seen at the 2
and 4% levels. Elevated PBI, due to interference by erythrosine with
PBI determinations rather than due to thyroid dysfunction, were seen.
T4 iodine levels were not affected. There were no other
haematological differences and no anaemia was seen. No adverse gross
pathology was noted; histopathology examinations did not show any
colour-related abnormalities (Hansen et al., 1973b).
Groups of 70 male and 70 female Charles River CD weanling rats
were fed erythrosine in the diet at levels of 0.1, 0.5, or 1.0% for 30
months after in utero exposure. Two concurrent control groups
(70 animals/sex/group) received no colour in the diet. The respective
average consumption of erythrosine was 49, 251, or 507 mg/kg b.w./day
for males and 61, 307, or 642 mg/kg b.w./day for females. There were
no consistent significant compound-related effects during the
in utero phase. In the main study, there were no consistent
significant compound-related effects on the following: physical
observation, behaviour, mortality, food consumption, haematology,
clinical chemistry, urinalysis, or ophthalmological findings. Mean
body weights of control and treated rats did not differ significantly
during the exposure period. The gross pathological changes that were
noted could not be attributed to treatment with erythrosine. The
incidence of non-neoplastic lesions was comparable between treated and
control groups. There was a statistically-significant increase in the
incidence of benign thyroid tumours (follicular adenomas): 6/68 in the
1.0% female test group versus 0/140 in the control group. The
incidence of malignant tumours in rats of treated groups was
comparable with that of the controls (Brewer et al., 1981).
Two groups of Charles River CD weanling rats (70 males and 70
females/group) were given erythrosine in the diet at dose levels of 0
or 4.0% for a period of approximately 29 months after in utero
exposure. The average consumption of the erythrosine was 2465 mg/kg
b.w./day for males and 3029 mg/kg b.w./day for females. There were no
consistent significant compound-related effects on the following;
physical observations, behaviour, mortality, food consumption,
haematology, clinical chemistry, urinalysis, or ophthalmological
findings. Mean body weights of treated rats (both sexes) were slightly
lower throughout the study than those of the control rats. These
differences were statistically-significant except at weeks 3-5 and 122
(males) and at weeks 0-4, 6, and 114 (females). The mean absolute and
relative thyroid weights of treated males were more than twice those
of the controls. Histopathological examination revealed that the
incidence of thyroid hyperplasia (follicular and C-cell) was
significantly increased in treated males. There was a statistically-
significant increase in the incidence of follicular adenoma of the
thyroid in treated male rats (16/68) when compared with the controls
(0/69). The incidence of malignant tumours, including thyroid C-cell
and follicular carcinomas, was comparable among treated and control
rats (Brewer et al., 1982).
Groups of 6-week-old pathogen-free F344 rats (50 males and 50
females) were fed diets containing erythrosine at levels of 1.25 or
2.5% for 18 months. The control group consisted of 30 males and 30
females that received a diet free of erythrosine. For the first 20
weeks of treatment, erythrosine was given in pelleted diet and for the
remaining treatment period it was given in powder diet. Rats exposed
to erythrosine were sacrificed 18 months, and the control rats 24
months, after the start of the study. No parameters other than
histopathology were reported. Histopathological examinations revealed
sporadic cases of spontaneous neoplasms (tumours of the genital
system, endocrine system, haematopoietic system, and digestive
system), but their frequencies were similar among animals in the
erythrosine-treated groups and they were comparable to the controls.
No pathological changes were observed in the thyroid glands
(Fukunishi et al., 1984).
A study was undertaken to investigate whether the thyroid tumours
found after chronic feeding of erythrosine to male rats at a dose
level of 4.0% in the diet resulted from excess iodine (either as a
contaminant of the colour or as iodine metabolized from the colour) or
from another non-iodine-related property of erythrosine. The study was
composed of the following six dose groups, each containing 70
(35 males and 35 females) Charles River CD rats, with continuous
exposure for 27 weeks:
Group 1 - unadulterated diet.
Group 2 - 80 µg of NaI (sodium iodide)/g of diet.
Group 3 - purified erythrosine at 4.0% in the diet.
Group 4 - purified erythrosine at 4.0% in the diet plus
80 µg of NaI/g of diet.
Group 5 - purified erythrosine at 4.0% in the diet plus
160 µg of NaI/g of diet.
Group 6 - commercial erythrosine at 4.0% in the diet.
The feeding of commercial erythrosine at a level of 4% in the
diet produced hyperthyroidism. TSH and T4 were elevated, while T3
concentrations were depressed. Changes in clinical chemistry
parameters, body weight, and food consumption were also indicative of
hyperthyroidism. Additional purification of the commercial preparation
of erythrosine to remove free iodide did not modify these effects.
These responses were not found after feeding a diet spiked with NaI
only. This study demonstrated that thyroid changes observed in this
and former studies are associated with increased TSH concentrations.
However, the mechanism for these effects of erythrosine cannot be
determined from the results of this study (Couch et al., 1983).
Twenty rats were subject to weekly s.c. injections of 1 ml of a
5% aqueous solution of erythrosine for 596 days (85 weeks). 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 s.c. with aqueous solutions of
erythrosine at 12 mg/animal once per week for 2 years. No tumours
either at the injection sites or in other parts of the body were
observed (Hansen et al., 1973b).
Gerbils
Three groups (15-16 animals/sex/group) of Mongolian gerbils,
approximately 6 months old, were fed diets containing erythrosine at
levels of 1.0, 2.0, or 4.0% for 105 weeks. Control groups (31 animals
of each sex) were fed diets free of erythrosine. Animals of all
treated groups exhibited a statistically-significant dose-related
decrease in body-weight gain when compared with the controls. In
general, there were slight, and in some isolated cases significant,
depressions of haematocrit and haemoglobin values and leucocyte and
reticulocyte counts in animals of treated groups. The relative weights
of heart, liver, and spleen were significantly decreased in animals of
both sexes at the two high-dose levels. Dose-related changes such as
enlargement of follicles and, in some cases focal hyperplasia, were
observed in the thyroids of treated animals. Histopathology did not
reveal any treatment-related effects (Collins & Long, 1976).
Groups of 20-24 male and Mongolian gerbils approximately 6 months
old received erythrosine (dissolved in water) by stomach intubation at
dose levels of 200, 750, or 900 mg/kg twice weekly for 97 weeks. A
control group (32 animals/sex) was intubated with distilled water
only. The dosages were administered in a volume of 10 ml/kg b.w. No
treatment-related adverse effects were observed for investigated
parameters such as clinical toxicity, mortality, body-weight gain,
haematology, organ weights, gross pathology, or histopathology
(Collins & Long, 1976).
Observations in man
Five human volunteers (four males and one female, aged
21-35 years) received erythrosine in the diet at dose levels of 5, 10,
or 25 mg/day in weekly increments for a period of 3 weeks. Total serum
iodine and PBI increased slowly and slightly in association with the
weekly increasing erythrosine doses. Other tests for serum T4, T3,
TSH, erythrosine, urinary iodine, erythrosine excretion, and
T3-resin uptake remained unchanged throughout the 3 weeks. Increases
in serum PBI and total serum iodine during the exposure period
indicates that a portion of the iodine ingested as erythrosine appears
to be absorbed from the gastrointestinal tract. No changes in
concentration of TSH, T4, and T3 in serum indicate that both the
thyroid function and thyroregulatory mechanisms were unaffected by the
ingestion of erythrosine during a three-week period at dose levels of
5, 10 and 25 mg/day in weekly increments (Ingbar et al., 1983).
Human volunteers were given single doses of 75-80 mg erythrosine
labelled with 131I in a milk-shake or lemonade; the subjects
received a daily dose of 5 drops of saturated potassium iodide
solution to block thyroid uptake of 131I. Whole-body counts were
carried out and complete stool and urine samples were examined for
activity; daily blood samples were also examined for total serum
131I, and for T4 and T3.
Faecal excretion of 131I approximated 100% in four subjects; in
two subjects, lower recoveries (80 and 90%) were achieved, but this
was probably due to incomplete collection, as the unrecovered activity
was not detectable in whole-body counts, urinary 131I, or serum.
Urinary excretion of label in 48 hours did not exceed 0.38% of the
dose in any subject and the urinary counts after 48 hours were similar
to background. Whole-body counts indicated that 131I-erythrosine was
eliminated rapidly and nearly completely, less than 1% of the
administered dose remaining after 7 days. The small amount of
remaining activity declined exponentially in the period from 7-14 days
after administration, with the half-life averaging 8.4 ± 2.1 days.
Extrapolation of this slow phase to zero time indicated a potential
initial body retention of 1.2 ± 0.4% of the administered dose.
Negligible quantities of 131I appeared in the serum, never exceeding
0.013% of the dose/l serum, and in none of the studies were the serum
T4 and T3 concentrations significantly altered following ingestion
of erythrosine.
These studies indicate that only a small proportion of ingested
erythrosine is absorbed from the gastrointestinal tract of man and, at
the single oral dose levels used, erythrosine did not affect thyroid
hormone levels (Ingbar et al., 1984b).
Comments
Further metabolic studies confirmed that erythrosine is only
absorbed to only a small extent from the gastrointestinal tract in
rats and man. Biochemical studies of thyroid function and of
metabolism of thyroid hormones in the liver and pituitary indicate
that erythrosine inhibits deiodination of T4 to T3 and,
consequently, at high dose levels, activates TSH secretory mechanisms
in the pituitary.
Morphological changes in the thyroid follicular cells induced by
prolonged exposure to high doses of erythrosine were reported to be
consistent with stimulation of synthetic and excretory processes and
to support the view that the development of thyroid tumours in
long-term studies in rats might be mediated by a hormonal effect. The
morphological changes were reported to be reversible by administration
of T3, even in rats continuing to be exposed to 4% erythrosine in
the diet, further supporting a hormonal mechanism.
Additional studies on the mutagenicity of erythrosine confirm
that it is non-genotoxic. No effects on thyroid function or regulatory
mechanisms were observed in human studies at dose levels up to 80 mg
(single dose) or in a three-week study in which erythrosine was
administered in incrementally-increased doses of 5, 10, and 25 mg
daily for 7 days.
EVALUATION
Level causing no toxicological effect
Rat: 0.25% in the diet, equivalent to 125 mg/kg b.w./day
(based on studies of biochemical effects on thyroid hormone metabolism
and regulation).
Estimate of temporary acceptable daily intake for man
0-0.6 mg/kg b.w.
Further work or information
Required (by 1988)
Parmacokinetic studies on erythrosine relating the amount of
absorption to the amount ingested, which would enable a correlation to
be established between blood/tissue levels of erythrosine and effects
on the thyroid.
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See Also:
Toxicological Abbreviations
Erythrosine (FAO Nutrition Meetings Report Series 46a)
Erythrosine (WHO Food Additives Series 6)
Erythrosine (WHO Food Additives Series 19)
Erythrosine (WHO Food Additives Series 24)
Erythrosine (WHO Food Additives Series 28)
Erythrosine (WHO Food Additives Series 44)
ERYTHROSINE (JECFA Evaluation)