INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
SUMMARY OF TOXICOLOGICAL DATA OF CERTAIN FOOD ADDITIVES
AND CONTAMINANTS
WHO FOOD ADDITIVES SERIES NO. 13
The data contained in this document were examined by the
Joint FAO/WHO Expert Committee on Food Additives*
Rome, 3-12 April 1978
Food and Agriculture Organization of the United Nations
World Health Organization
* Twenty-second Report of the Joint FAO/WHO Expert Committee on Food
Additives, Geneva, 1978, WHO Technical Report Series No. 631
AZORUBINE
Explanation
This colour was evaluated at the Joint FAO/WHO Expert Committee
on Food Additives in 1974. Since the previous evaluation, additional
data have become available and are summarized below.
BIOLOGICAL DATA
Acute toxicity
Acute testing of azorubine administered by various routes has
resulted in the findings summarized below:
Species Route LD50 References
Rat i.p. 1 g/kg Gaunt et al., 1967
Mouse i.p. 0.8 g/kg Gaunt et al., 1967
Mouse i.v. 0.8 g/kg Deutsche Forschung, 1957
Mouse Oral >8 g/kg Gaunt et al., 1967
Rat Oral >10 g/kg Gaunt et al., 1967
Short-term studies
Rat
Sixteen Carworth Farm E strain rats of each sex were placed into
groups which were fed 0.0, 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 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.,
1977a).
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 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 was that
azorubine was non-carcinogenic in mice (Bonser et al., 1956).
Azorubine was administered to ASH/CSI 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 were fed only the
basal ration of Oxoid pasteurized diet supplement with 80 ppm 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 these same values decreased. The 1.25%
females were also found to have a decreased packed cell volume at week
52. No abnormal tumour 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 al., 1974).
Rat
Azorubine was fed at levels of 0.0, 0.35, 0.8 and 2.0% of the
diet to Sprague-Dawley rats (30 male and 30 female 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 of up to 2% for 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., 1977a).
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, and gross 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 al., 1977b).
Miscellaneous studies
Female New Zealand white rabbits were administered azorubine on
days 6 through 18 of gestation by oral intubation at levels 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).
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 al., 1972b).
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 succinic oxidase system of rat
liver homogenates. The results indicated that this dye inhibited the
oxidative activity of this enzyme approximately 40% (Sikorska and
Krauze, 1962).
Administration of azorubine at doses up to 10 mg/kg produced no
alterations of the blood pressure of anaesthetized dogs and rabbits
(Vrbovsky and Selecky, 1959).
Commercial azorubine preparations were fed to bull calves at
levels of 1, 5, and 10 g/100 kg of skim milk powder. No dye was
detected in any of the meat samples analysed, although the time from
administration to analysis is not known (DeMoor et al., 1969).
REFERENCES
Smith, J. M., Kasner, J. A. and Cannelongo, B. (1972a) Carmoisine,
Segment II. Rabbit Teratology Study. Bio/dynamics, Inc. Project No.
71R-721F submitted to Inter-Industry Color Committee: Cosmetic,
Toiletry and Fragrance Association. Unpublished report
Smith, J. M., Kasner, J. A. and Andresen, W. (1972b) Carmoisine,
Segment II. Rat Teratology Study. Bio/dynamics, Inc. Project No.
71R-719F submitted to Inter-Industry Color Committee: Cosmetic,
Toiletry and Fragrance Association. Unpublished report
Bonser, G. M., Clayson, D. B. and Jull, I. W. (1956) The induction of
tumors of the subcutaneous tissues, liver, and intestine in the mouse
by certain dyestuffs and their intermediates, Brit. J. Cancer, 10,
653-667
DeMoor, H., Hendrickx, H., Casteels, M., Eekhout, W. and Buysse, F.
(1969) Azorubine in commercial products and its presence in meat,
Meded. Rijksfac. Landbouwwetensch, 34(4), 1067-1079 (NETH)
Deutsche Forschungsgemeinschoft-Farbstoff-Kommission (1957)
Mitteilung, 6, 2. Auflage Toxikologische Daten von Farbstaffen
und ihre zullassung fur Lebensmittel in verschiedenen Landern,
p. 38, Franz Steiner Verlag GMBH, Wiesbaden
Gaunt, I. F., Farmer, M., Grasso, P. and Gangolli, S. D. (1967) Acute
(mouse and rat) and short-term (rat) toxicity studies on carmoisine,
Food Cosmet. Toxicol., 5, 179-185
Gaunt, I. F., Grasso, P., Kiss, I. S. and Gangolli, S. D. (1969)
Short-term toxicity study on carmoisine in the miniature pig,
Food Cosmet. Toxicol., 7, 1-7
Holmes, P., Pritchard, A. B., Kirschman, J. C., Sefecka, R. and Ford,
A. (1977a) Multigeneration reproduction studies with carmoisine in
rats, submitted for publication
Holmes, P. A., Pritchard, A. B., Kirschman, J. C., Sefecka, R. and
Ford, A. (1977b) A one year feeding study in rats, submitted for
publication
Mason, P. L., Gaunt, I. F., Butterworth, K. R., Hardy, J., Kiss, I. S.
and Grasso, P. (1974) Long-term toxicity studies of carmoisine in
mice, Food Cosmet. Toxicol., 12, 601-607
Sikorska, E. A. and Krauze, S. (1962) Wplywniektorych barwnikow
zywnosciowych i kosmetycznych na aktywnosc oksydazy bursztynianowej,
Roczniki Pzh., t XIII, No. 5, 457-466
Vrbovsky, L. and Selecky, F. V. (1959) Niektore vitalne a ine farbiva
ovplyvnujuce krvny tlak, Bratisl. lek. Listy, 39, 737-752
See Also:
Toxicological Abbreviations
Azorubine (WHO Food Additives Series 6)
Azorubine (WHO Food Additives Series 18)
AZORUBINE (JECFA Evaluation)