AZORUBINE
Explanation
This colour was evaluated for acceptable daily intake by the
Joint FAO/WHO Expert Committee on Food Additives in 1974 and 1978 (see
Annex I, Refs. 34 and 48), and tentative specifications and a
toxicological monograph were prepared in 1977 and 1978 respectively
(see Annex I, Refs. 45 and 49). Since the previous evaluation,
additional data had become available and are summarized and discussed
in the following monograph. The previous monograph has been expanded
and is reproduced in its entirety below.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Absorption, distribution, excretion and metabolism
Male Swiss albino mice (CD-1) (three to six per group) were given
single doses of (14C)azorubine (5 µCi/mmol) by stomach tube
(200 mg/kg, 6 µCi) or i.v. injection (200 mg/kg, 0.7 µCi). The plasma
and tissue kinetics of the compound were studied by monitoring the
decay of radioactivity in plasma, gastrointestinal tract, liver,
kidney, lung, testes, spleen and gall bladder, 5, 10, 15, 30 minutes
and 1, 2, 4, 8, 16, 32 and 96 hours after dosing. The faeces and urine
of mice placed in individual metabolic cages were collected between
four and 96 hours after dosing. After oral administration, peak levels
of radioactivity occurred in plasma (0.08%/ml) and in the liver, lung,
testes and spleen eight hours after dosing. Radioactivity was almost
completely excreted in faeces (74%) and urine (19%) within 16-32 hours
of oral dosing. After i.v. injection of (14C)azorubine, most of the
radioactivity (76%) was excreted 24 hours after dosing in faeces (64%)
and urine (12%). The plasma 14C-radioactivity decay curve after i.v.
administration indicated a very rapid distribution of the compound
into the tissues (t1/2 = 10 minutes) and an efficient excretion
mostly through the gastrointestinal tract (92%) which was complete 48
hours after dosing (Galli et el., 1981).
Rats were injected intravenously with approximately 1 mg of the
dye. The bile was collected for six hours and analysed. The recovery
of the dye was an average of 38% (30-40%) of the administered quantity
(Ryan & Wright, 1961). This like any other azo dye is probably reduced
in the gut by bacterial azo reductases (Walker, 1970). The absorption,
distribution and excretion of the red azo dye azorubine were studied
in male Sprague-Dawley rats. (14C)azorubine (5 mCi/mmol) was
administered to groups of at least three animals in a dose of
200 mg/kg (25 µCi) by gavage or in the same dose (200 mg/kg, 3 µCi) by
intravenous injection and radioactivity was measured in blood, tissue,
faeces and urine at different times after dosing (5, 10 and 30
minutes and 1, 2, 4, 8, 16, 32, 64 and 96 hours after azorubine
administration). After oral administration of the dye, no
radioactivity was detected in the brain, adipose tissue, muscle,
testes, spleen or lung, and recovery of the administered radioactivity
in faeces and urine was almost complete by 32 hours (82% and 8%
respectively). The radioactivity profile of the blood indicates rapid
but poor absorption of (14C)azorubine, a maximum radioactivity
content, corresponding to 0.01% of the dose per ml of blood, being
reached within 10 minutes. The decay curve for 14C-radioactivity in
the blood after i.v. injection of (14C)azorubine indicated rapid
distribution to the tissues and could be described in terms of a two-
compartment mathematical study. The highest levels of radioactivity
occurred in the gastrointestinal tract and liver after the injection,
but after 24 hours no radioactivity was detectable in these or other
tissues. All the radioactivity was recovered in the faeces and urine
in the 24 hours following i.v. injection, the 79% of the dose present
in the faeces indicating active excretion of the dye and its
metabolites in the bile and poor reabsorption from the intestine. The
bioavailability of (14C)carmoisine, calculated from the blood-
radioactivity curves after oral and i.v. administration, was less than
10% (Galli et el., 1982a).
The absorption, metabolism and excretion of orally administered
(14C)-labelled azorubine (32 mCi/mmol) have been studied in male and
female Wistar albino rats, male MF-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), the majority of the radioactivity
was excreted in the urine and faeces in the first 24 hours: 18% and
73% in rats, 17% and 66% in mice, and 37% and 45% in guinea-pigs
respectively. Less than 0.03% of the dose was eliminated as CO2.
Substantially all of the dose was recovered in the excrete within
72 hours, the majority being accounted for in the faeces. Although the
male and female rat and the mouse excreted a similar proportion of the
dose in the urine, the proportion of the radioactivity found in the
urine of the guinea-pig was significantly greater than that of the
other species at both dose levels. Pretreating male rats with
unlabelled colouring in the diet (0.05% w/w) for 28 days to provide an
intake of approximately 50 mg/kg/day prior to dosing with
14C-labelled colouring (50 mg/kg), had no effect on the route of
excretion or the time taken to eliminate all of the label, although
there was evidence that the proportion of the metabolites extracted
from the faeces was different from the corresponding untreated
animals. Following a single dose of 14C-labelled colouring to non-
pretreated 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 at 72 hours.
Pregnant rats eliminated a single oral dose of 14C-labelled colouring
(50 mg/kg at day 8 of pregnancy) at a similar rate to non-pregnant
females, and the concentration of radioactivity in the foetuses was
similar to that in the other tissues. Examination of urine by high-
performance liquid chromatography showed that between 60% and 80% of
the radioactivity in the urine was associated with naphthionic acid in
the urine of all three species. A further 10% and 20% of the
radioactivity in the urine co-eluted with 2-amino-1-naphthol-4-
sulfonic acid (2-ANS). The third component, accounting for less than
5% of the radioactivity in the rat and mouse but 16% in the guinea-pig
co-chromatographed with 1,2-NQS (1,2-naphthoquinone-4-sulfonate) and
the fourth, which accounted for between 2% and 5% of the radioactivity
in the urine, was not identified. Naphthionic acid was also found in
the faeces of all three species; however, no 2-ANS or 1,2-NQS was
detected. Five unidentified metabolites were found in the faeces of
all three species, the proportions of which varied between species.
Two of these metabolites were hydrolysed by combined ß-glucuronidase
and sulfatase treatment. No significant absorption of radioactivity
during a one-hour period was found from isolated loops of small
intestine of the rat, mouse or guinea-pig containing either 50, 500 or
5000 ppm (0.005, 0.05 or 0.5%) carmoisine as measured by total
recovery of injected radioactivity. Less than 0.03% of the
administered radioactivity in the 50 mg/kg dose was recovered in the
bile during one hour and only between 0.04% and 0.7% during five hours
(Phillips et al., 1982).
(14C)azorubine was administered to rats at the dose of 200 mg/kg
bw (25 µCi) by gavage. Separation of radioactive compounds in faeces
and urine of animals was carried out by HPLC with a UV and a
radioactivity detector. In addition to unmodified carmoisine, five
radioactive compounds were present. The main peak showed both the
retention time and UV spectrum of authentic naphthionic acid.
Metabolic patterns similar to those observed in vivo were found by
incubation of 14C-carmoisine under anaerobic conditions with a
bacterial suspension isolated from human faeces and from the
intestinal contents of rats (Marinovich et al., 1983).
Effects on enzymes and other biochemical parameters
In vitro assays were conducted by inclusion of azorubine
(0.4 mg/mg tissue) in enzyme activity trials in an attempt to
determine the effects of the dye on the succinic oxidase system of rat
liver homogenates. The results indicated that this dye inhibited the
oxidative activity of this enzyme by approximately 40% (Sikorska &
Krauze, 1962).
TOXICOLOGICAL STUDIES
Special studies on carcinogenicity
Two batches of textile grade azorubine (71.4% dye, 7.39% water,
11.70% NaC1, 5.70% Na2SO4, 3.72% NaHCO3 for the first 11 months
and 67.30% dye, 7.48% water, 7.85% NaC1, 12.20% Na2SO4, 5.16%
NaHCO3 for the final 13 months) were used to conduct a
carcinogenicity bioassay in mice and rats. Groups of mice (50 males
and females per group) were fed diets containing textile grade
azorubine to study potential carcinogenicity effects at levels of 0,
3000 or 6000 ppm (0, 0.3 or 0.6%) for 103-104 weeks. Throughout the
study, mean body weights of dosed female mice were comparable with
those of the controls, while the mean body weight of high-dose male
mice was slightly lower than that of the controls. No other compound-
related clinical signs were observed. There was no significant
decrease in survival between any of the groups of male or female mice.
Results of histopathological examination indicated that administration
of azorubine to male and female mice under the conditions of the
bioassay was not associated with an increased incidence of any rumour
type. However, although not dose related, non-neoplastic lesions as
lymphoid hyperplasia of the spleen, haematopoiesis in the liver and
lymphoid hyperplasia of the submucosa of the urinary bladder were
observed in female mice (Anon., 1982).
Textile grade azorubine was administered in diets containing 0,
6000 or 12 500 ppm (0, 0.6 or 1.25%) of the dye for 103-104 weeks to
groups of 50 male F344 rats, and 0, 12 500 or 25 000 ppm (0, 1.25 or
2.5%) to groups of 50 female F344 rats. Control animals (90) were
shared with feeding study of CI Acid Orange 10 and FD and C Yellow
No. 6, which were conducted concurrently. Mean body weights of dosed
rats of either sex were comparable with those of the controls
throughout most of the study. No compound-related clinical signs were
observed. The survival of the low-dose group of male rats was
significantly greater than that of the controls (P = 0.046) or of the
high-dose group (P <0.001). No significant differences were observed
between the control and high-dose groups of male rats or between any
groups of female rats. Endometrial stromal polyps of the uterus were
observed in high-dose female rats at an incidence significantly higher
(P = 0.008) than that seen in the controls (controls: 9/87, 10%; low
dose: 11/50, 22%; high dose: 14/50, 28%). However, the observed incidence
of polyps in the dosed groups was similar to the historical rate in
untreated female F344 rats (65/286, 23%; range 10-37%). Hence, the
increased incidence of this lesion is not regarded as being associated
with the administration of azorubine. The various non-neoplastic lesions
represented among both control and dosed animals have been encountered
previously as spontaneous occurrences in aging laboratory rats. An
increased incidence of adrenal cortical focal hyperplasia,
characterized by focal collections of basophilic, eosinophilic or
vacuolated cells, was seen in high-dose rats of both sexes (males:
5/89, 6%; 6/49, 12%; 8/50, 16%; females: 7/86, 8%; 7/50, 14%; 18/50,
36%). Results of histopathological examination indicated that
azorubine was not carcinogenic to male or female F344 rats under the
conditions of this bioassay (Anon., 1982).
Special studies on Heinz bodies
Four cats were given 5% aqueous solution in doses of 1 g on the
first day and 0.1 g on the ninth and eighteenth days. A negative test
for Heinz bodies was obtained (Deutsche Forsch., 1957).
Special studies on mutagenicity
The colour was tested for mutagenic action in a concentration of
0.5 g/100 ml in cultures of Escherichia coli. No mutagenic effect
was found (Lück & Rickerl, 1960). Testing of azorubine for
mutagenicity with Salmonella typhimurium TA-1538 50 µg/plate showed
that the azo dye is not mutagenic in the absence or presence of a
liver enzyme preparation (Garner & Nutman, 1977). This colour was
tested for cytotoxic activity and for mutagenic effect in a
concentration of 0, 1, 2, 20, 500 and 1000 µg/plate/108 bacteria in
cultures of different strains of Salmonella typhimurium TA-1535,
TA-1538, TA-100 and TA-98 either in the presence or absence of liver
microsomal fraction. No mutagenic effect was found (Viola & Nosotti,
1978). Azorubine 5 mg/ml did not induce mitotic gene conversion in
Saccharomyces cerevisiae BZ 34 when treated either in stationary-
phase or log-phase culture without microsomal activation. Under these
treatment conditions neither significant cell killing nor inhibition
of cell division was observed (Sankaranarayanan & Murthy, 1979). No
evidence of mutagenic potential of azorubine (5 mg/ml) was obtained in
two different bacterial test systems, Escherichia coli WPZ and
Salmonella typhimurium TA-1538, either in the presence or absence of
liver microsomal fraction (S-9 mix) (Haveland-Smith & Combes, 1980).
Special studies on placental transfer
Three or six pregnant Sprague-Dawley rats per group received
(14C)azorubine (200 mg/kg, 25 µCi) by gavage on days 16-19 of
gestation. Animals were killed on day 19 of gestation, two, 16 and
64 hours after dosing, and blood, maternal tissues, amniotic fluids,
placentae, maternal uterus, foetal membranes and foetuses were
analysed for radioactivity. No evidence for transplacental transfer of
(14C)azorubine or its metabolites was obtained. More than 90% of the
radioactivity was excreted in faeces and urine within 64 hours. In a
similar experiment no significant differences in maternal body weight,
food intake of dams, number of foetuses, litter size and foetal weight
were observed in treated (200 mg/kg, 25 µCi) and control animals when
the dye was administered by gavage on day 11 of gestation, and the
animals were killed on day 19 of gestation (Galli et el., 1982b).
Special studies on reproduction
Rat
Twenty-five male and 25 female rats received 1% azorubine in
their drinking-water for 180 days, giving approximately 55 g dye per
animal. A similar group of 50 rats acted as controls. Weight gain,
mortality and general condition were similar in both groups. After
seven months the animals were mated and an F1 generation produced.
After weaning the pups were put again on 1% azorubine and after four
months mated to produce an F2 generation. No abnormalities regarding
litter number or fertility were noted. After 200 days on 1% azorubine,
the F2 generation was kept on normal diet and water for two years. No
adverse effects were seen on mortality or tumour incidence (Hecht,
1966).
Special studies on sensitizing effects
In experiments on guinea-pigs, it was found that this colour had
no sensitizing activity (Bär & Griepentrog, 1960).
Special studies on teratogenicity
Female Long-Evans rats were administered azorubine at levels of
100 mg/kg/day (22 animals), 300 mg/kg/day (24 animals), and 1000
mg/kg/day (22 animals) on days 6-15 of gestation by oral intubation.
Sixty-six rats served as control animals receiving the methylcellulose
(0.5%) vehicle, and 22 animals were dosed with 30 mg/kg/day of trypan
blue as a positive control. No embryotoxic or teratogenic effects were
seen in the animals administered azorubine (Smith et el., 1972b).
Female New Zealand white rabbits were administered azorubine on
days 6 through 18 of gestation by oral intubation at a level of 0 (47
animals), 40 mg/kg/day (15 animals), 120 mg/kg/day (15 animals) and
400 mg/kg/day (20 animals) in a teratology study. Thalidomide
(150 mg/kg/day) was administered to 15 rabbits as a positive control.
Of the dye-treated animals, no effect was seen on body weight gain. A
statistically non-significant increase in the number of spontaneous
deaths among dams of the high-dose group was found to be present.
There was also a decrease in the implantation efficiencies of all
females to which azorubine had been administered. This, however, was
not deemed to be compound related in that implantation was assumed to
have occurred prior to the initiation of the dye administration. No
signs of toxicity or foetal abnormalities were found, thereby
indicating that azorubine, at the levels administered, is non-
teratogenic (Smith et al., 1972a).
Acute toxicity
Acute testing of azorubine administered by various routes has
resulted in the findings summarized below:
Animal Route LD50 Reference
(g/kg bw)
Mouse i.p. 0.8 Gaunt et al., 1967
i.v. 0.8 Deutsche Forsch., 1957
oral > 8.0 Gaunt at al., 1967
Rat i.p. 1.0 Gaunt et al., 1967
oral >10.0 Gaunt et al., 1967
Administration of azorubine at doses up to 10 mg/kg produced no
alterations of the blood pressure of anaesthetized dogs and rabbits
(Vrbovsky & Selecky, 1959).
Short-term studies
Rat
Three weanling rats were given a 0.1% solution of carmoisine to
drink for 28 days (daily consumption approximately 15 mg). No toxic
effects were noted (Goldblatt & Frodsham, 1952). Sixteen Carworth Farm
E strain rats of each sex were placed into groups which were fed 0,
0.05, 0.10, 0.50 and 1.0% azorubine for 90 days. Feeding of this
colour at these levels produced no deleterious effects on body weight,
food consumption, haematology, renal or hepatic function parameters.
Females at the 1.0% dietary level were found to have elevated renal
weight, but no untoward pathology was found upon examination of this
organ. No non-spontaneous, compound-induced tumours were found and no
abnormal gross pathology was observed. A no-effect level of 0.5%
(250 mg/kg/day) has been established for rats in a 90-day study, based
upon the elevated female renal weights (Gaunt et al., 1967). Sprague-
Dawley rats (10/sex/experimental group; 20/sex/control group) were fed
0, 2, 4, 6 or 8% azorubine in the basal ration of Wayne Lablox for
nine weeks. At levels of 6.0% or greater, the toxic effect elicited by
this colour was seen to be a reduction in body weight gain of animals
in these groups. No other toxic manifestations were noted. This
equates to a no-effect level of 2000 mg/kg (Holmes et al., 1978a).
Pig
Three male and three female Pitman-Moore crossed Palouse strain
miniature pigs per group were administered azorubine at levels of 0,
250, 500 and 1000 mg/kg/day admixed with a basal diet composed of
Hi-lean Rearers Pencils for 90 days. No untoward toxicology or
pathology was noted at the conclusion of this study and no significant
differences between control and treated animals were detected. A
no-effect level of 1000 mg/kg/day was assigned based upon the results
of this study (Gaunt et al., 1969).
Long-term studies
Mouse
Thirty mice (15/sex) were administered azorubine subcutaneously
for 52 weeks. The initial dose consisted of 0.1 cc of a 3% solution of
the colour in arachis oil two times per week, which was increased to
6% at the end of six months. Control mice received the arachis oil
diluent alone in subcutaneous injections. Following the 52-week
administration period, at which time each animal had received 468 mg,
the animals were allowed to survive as long as possible. At the end of
89 weeks after the initiation of the treatment, one male and 11 female
mice had expired. Seven of the females had been found to develop
lymphomas, while no subcutaneous sarcomas or hepatomas were observed.
The lymphosarcomas observed were also seen to develop spontaneously in
control animals and no toxicological significance was imparted to
those observed. The conclusions drawn by the authors were that
azorubine was non-carcinogenic in mice (Bonser et el., 1956).
Azorubine was administered to ASH/CS1 strain male and female mice
(30/sex/group) for 80 weeks at levels of 0.01, 0.05, 0.25 or 1.25% of
the diet. A control group of 60 animals per sex was fed only the basal
ration of Oxoid pasteurized diet supplemented with 80 ppm (0.008%)
vitamin K3 and water ad libitum. The feeding of diets containing
the colour additive had no effect on the behaviour, body weight or
organ weight of the animals entered into the study. Female mice fed
1.25% were found to possess significantly lowered (P <0.001)
haemoglobin levels at weeks 12 and 52 of the study and, at week 52,
males fed 0.25% and 1.25% dye were found to have a decreased packed
cell volume. No abnormal rumour distribution which could be considered
to be compound related was detected. The minimum toxic effect level
seen was 1.25%, with the symptoms being mild anaemia at week 80. A
no-effect level of 0.25% (375 mg/kg/day) was ascribed to azorubine fed
to mice over a period of 80 weeks (Mason et at., 1974).
Rat
Ten rats were given the colour in the drinking-water in a
concentration of 1% for 209 days. The daily intake was 1.2 g/kg bw and
the total amount administered was 51 g per animal. The observation
period was 919 days. No tumours were found (Deutsche Forsch., 1957).
Ten rats were given 1% of the colour in the drinking-water for 250
days. The daily intake was 7.94 g/kg bw and the total intake 52 g per
animal. The observation period was 545 days. No tumours were found
(Deutsche Forsch., 1957). Ten rats were given a diet containing 0.2%
of the colour for 417 days. The daily intake was approximately
0.1 mg/kg bw and the total intake was 11 g per animal. The observation
period was 838 days. No tumours were found (Deutsche Forsch., 1957). A
group of 10 rats were given twice weekly subcutaneous injections of
0.5 ml of a 1% solution (= 5 mg) of the colour for one year. The
animals were kept under observation for over 938 days. One axillary
tumour was observed in one animal (Deutsche Forsch., 1957). In a
repeat experiment another group of 10 rats was given twice weekly
subcutaneously 0.5 ml of a 1% solution (= 5 mg) of the colour for one
year. No tumours were formed after 521 days, each animal having
received 0.5 g (Deutsche Forsch., 1957).
Azorubine was fed at levels of 0, 0.35, 0.8 and 2.0% of the diet
to Sprague-Dawley rats (30 males and 30 females per group; 50 rats of
each sex in the control group) in a multigeneration reproduction
study. No deleterious effects were seen in the reproductive parameters
assessed (fertility, viability and lactation indices). No effects on
body weight gain were observed although, with each successive
generation, there was a trend toward increased dye consumption, this
being indicative of increased food consumption. Thus, azorubine had no
adverse effects on viability and reproductive abilities of rats when
fed at levels up to 2% in a study which included three in utero
exposures of the subsequent generations, as seen in the F0, F1a,b, F2a,b
and F3a,b generations (Holmes et al., 1978a). Thirty male and 30
female Sprague-Dawley rats, delivered following two generations of
parental in utero (F3b) exposures to azorubine, were placed into
groups to receive 0.35, 0.8 and 2.0% of this colour additive for one
year. The control group consisted of 50 animals of each sex. No
adverse, dye-related effects on body weight gain were observed. A
statistically significant increase (P <0.01) in bronchitis and
tracheal irritation was found in male rats fed azorubine at a level of
2.0% of the diet. Urinalysis, other haematological values, gross
pathological and histopathological findings were within normal limits.
A no-effect level of 0.8% (400 mg/kg/day) was assigned for azorubine
in rats, although the authors believed that the true value would have
been higher (Holmes et el., 1978b).
Groups of 114 (control) and 66 (treated) Wistar rats of each sex
were given a diet to provide intakes of 0 (control), 100, 400 or
1200 mg azorubine/kg/day for nine weeks (F0 generation). Diet
composition was:
Dye content 89.5%
Volatile matter 4.7%
Sodium chloride 4.7%
Sodium sulfate 1.7%
These animals were mated and the females allowed to rear the resulting
offspring, with the treatment continuing throughout mating, pregnancy
and lactation. Young were randomly selected to provide groups of 90
(control) and 54 (treated) of each sex. These were given the same
treatment as their parents for up to 110 weeks for the females or 115
weeks for the males (F1 generation). The appearance of the rats was
normal apart from an external contamination of the fur, colour in the
urine and dark faeces. Animals of both generations given 1200 mg
azorubine/kg/day were slightly lighter than the controls, despite a
small increase in food intake. There was an increased water intake by
these same animals and, on the basis of periodic renal function tests,
a tendency to excrete larger volumes of urine. The ability of the
kidney to concentrate urine under condition of dehydration was not
impaired. Haematological analysis on 20 animals of each sex at 3, 6,
12, 18 and 24 months and on all survivors at the end of the study
revealed isolated statistically significant differences between the
treated and control rats, but these were not consistent between the
sexes or with time and were not considered to be related to the
treatment. There were increases in caecum weight at the highest dose
level, but no other changes in organ weights that were due to
treatment. Investigations of kidney function using 20 animals of each
sex at 3, 6, 9, 12, 18 and 24 months did not reveal any changes that
could be related to azorubine treatment. There were low concentrations
of glucose in serum collected from the survivors of both sexes given
1200 mg azorubine/kg/day and females given 400 mg/kg. In the absence
of any associated findings, this could not be firmly related to
treatment.
There were a few high-dose males with bladder hyperplasia
possibly due to irritant metabolites in the urine. There was an
increase in the number of high-dose females with adrenal blood/fibrin
cysts and five high-dose females with intimal hyperplasia/medial
hypertrophy of the pancreatic blood vessels. The incidences of most
rumours were similar in treated and control rats. There was a small
increase in the number of adrenal phaeochromocytoma in the high-dose
males, but the incidence was well within the background for the same
strain of rat (Stevenson et al., 1982).
Comments
Following administration of a single dose of (14C)azorubine to
male and female rats, mice and guinea-pigs, the majority of the
radioactivity was excreted in the urine and faeces in the first
24 hours. Substantially all of the dose was recovered in the exereta
within 72 hours, the majority being accounted for in the faeces
(60-75%). Following a single dose of 14C-labelled colouring to non-
pretreated rats, mice and guinea-pigs or rats given repeated doses of
unlabelled colouring, no marked accumulation of radioactivity in any
tissue was found. Pregnant rats eliminated a single oral dose of
14C-labelled colouring at a similar rate to non-pregnant females and
no evidence for transplacental transfer of (14C)azorubine or its
metabolites was obtained. Naphthionic acid was found in the urine
(60-80% of the radioactivity) and faeces of all three species. A
further 10-20% of the radioactivity in the urine was co-chromato-
graphed with 2-amino-1-naphthol-4-sulfonic acid and less than 5% of
the radioactivity in the rat and mouse, but 16% in the guinea-pig
co-chromatographed with 1,2-naphthoquinone-4-sulfonate. Five
unidentified metabolites were found in the faeces of all three
species. No evidence of mutagenic potential of azorubine was obtained
in different bacterial tests systems in the presence of absence of
liver microsomal fraction. A carcinogenic bioassay of textile grade
azorubine was conducted in rats and mice fed up to 1250 and 900 mg/kg
bw, respectively. Under the conditions of this bioassay, textile grade
azorubine was not carcinogenic for rats or mice of either sex.
Reproduction studies including teratogenicity did not reveal any
compound-related adverse effect. A long-term study carried out in the
mouse indicates a no-effect level of 0.25% (375 mg/kg/day). A
multigeneration reproduction study in the rat did not show adverse
effects in the reproductive parameters assessed, in body weight gain,
in urine and haematological values, in gross pathological and
histopathological findings. An adequate one-year long-term study in
rats exposed in utero to azorubine indicates that there were not
changes that could be related to treatment. With the exception of the
higher caecum weights at the highest dose, the remaining organ weights
were not influenced by azorubine. The histopathology did not reveal
any significant differences between the control and treated animals.
There was no evidence of a treatment-related increase in the total
number of animals with benign and malignant rumours. It is concluded
that azorubine is not carcinogenic and that the no-untoward-effect
level in this study was 400 mg/kg bw per day of azorubine.
EVALUATION
Level causing no toxicological effect
Mouse: 0.25% (2500 ppm) in the diet, equivalent to 375 mg/kg bw.
Rat : 0.8% (8000 ppm) in the diet, equivalent to 400 mg/kg bw.
Pig : 0.1% (1000 ppm) in the diet, equivalent to 400 mg/kg bw.
Estimate of acceptable daily intake for man
0-4.0 mg/kg bw.
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in der Lebensmitteln, Med. u. Ernahr., 1, 99-104
Bonser, G. M., Clayson, D. B. & Jull, I. W. (1956) The induction of
tumors of the subcutaneous tissues, liver and intestine in the
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See Also:
Toxicological Abbreviations
Azorubine (WHO Food Additives Series 6)
Azorubine (WHO Food Additives Series 13)
AZORUBINE (JECFA Evaluation)