CARMINES
Explanation
Cochineal, carmines and carminic acid were last evaluated for
acceptable daily intake for man by the Joint FAO/WHO Expert Committee
on Food Additives in 1981 (see Annex I, Ref. 57).
Since the previous evaluation, additional data have become
available and the previous monograph has been expanded.
Cochineal carmine is obtained from aqueous extracts of cochineal,
which consists of the dried bodies of the female insect Dactylopius
coccus Costa. The colouring principle of carmines is the hydrated
aluminium chelate of carminic acid, in which aluminium and carminic
acid are thought to be present in the molar ratio 1:2 (Meloan et al.,
1971).
In commercial products the colouring principle is present in
association with ammonium, calcium, potassium or sodium cations,
singly or in combination, and these cations may also be present in
excess. Ammonium carmines exhibit solubility over a wide range of pH
while calcium carmines are sparingly soluble at pH values below 7.
Commercial products also contain proteinaceous material derived from
the source insect, and may contain free carminate anion or small
excesses of aluminium cations (Lloyd, 1980).
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
No information on metabolism is available.
TOXICOLOGICAL STUDIES
Special studies on mutagenicity
Carminic acid was negative in the Bacillus subtilis rec-assay
for DNA-damaging ability (Kada et al., 1972).
Carminic acid was not mutagenic for several strains of
Salmonella typhimurium in the presence of liver microsomal
preparations or enzymatic extracts of rat caecal microflora (Brown &
Brown, 1976; Brown el al., 1977).
Carminic acid did not produce reverse mutations in 4 strains
(TA 1535; TA 1537; TA 98; TA 100) of Salmonella typhimurium when
tested in presence and absence of liver microsomal (S9) fractions
obtained from animals pretreated with phenobarbitone. There was no
evidence of gene conversions when carminic acid was tested similarly
in vitro with Saccharomyces cerevisiae D strain, nor of forward
mutations in vitro or in vivo in host mediated assay using
Schizosaccharomyces pombe (Barale et al., 1978). Similar results
have been obtained in studies using Salmonella typhimurium TA 1538
and Escherichia coli WP2 uvr A (Haveland-Smith & Combes, 1980).
Special studies on skin sensitization
Three subjects with lip lesions gave positive patch tests when
tested with red lip salve containing calcium carmine, but negative
reactions to colourless lip salve. Since the coloured product had
previously been used by each individual, the symptoms of allergic
sensitivity were ascribed to carmine (Sarkany et al., 1961).
Special studies on teratogenicity
The embryotoxicity and teratogenicity of carmine have been
studied in mice. Mice were killed on day 19 of gestation, after i.p.
injection of lithium carmine or sodium carmine on day 8. Treated
animals of both groups showed resorption rates (20%) higher than those
of control groups (2%). The malformation rate was about 16% in the
lithium carmine group and 2.5% after injection of sodium carmine. Only
animals given sodium carmine showed an increase in the number of
retarded foetuses (Schluter, 1970).
Groups of mice were injected once with 2.5% lithium carmine at a
dose of 150 mg carmine/kg on days 6, 8, 10, 12 or 14 of pregnancy. A
teratogenic effect was observed on the first 3 treatment days, with
the maximum effect on day 8 (Schluter, 1971a,b).
Four groups of 30 mated female rats were given daily 0, 200, 500
or 1000 mg/kg bw of ammonium carmine by gastric intubation as aqueous
solution during pregnancy days 0 to 20. A group of 17 similar animals
received a solution of chlorides to provide an intake of sodium,
potassium and ammonium ions equal to that resulting from the highest
dose level of carmine. No adverse effects were noted on body weight,
pregnancy rate, pre-implantation losses, the average number of live
young litter weight or foetal weight. The group given the highest dose
of carmine and the cations control had an increased number of
implantation sites and of post-implantation losses. The latter was
considered to be due to an inability to maintain the increased number
of implantations rather than to an embryotoxic effect. No teratogenic
effects were noted in the foetuses and the degree of ossification of
those from the carmine-treated rats tended to be more advanced than
those from the control (Gaunt et al., 1976).
Special studies over 3 generations
Ammonia carmine was administered to Wistar rats over several
generations at dietary concentrations designed to provide intakes of
0, 50, 150 or 500 mg/kg bw per day. Animals of both sexes were used in
groups of 36 for treatment with carmine and groups of 60 for the
control. After a suitable period of treatment, the original animals
(generation F0a) were mated to provide generation F1a and then
remated to produce generation F1b. Generation F1a animals provided
animals for generation F2, which in turn provided the final F3
generation.
There were no effects on body weights, food and water intakes,
fertility or organ weights in adults of generations F0a, F1a or F2
which could be attributed to treatment. Post mortem examinations and
organ weight measurements of pups of generations F1b, F2 and F3 did
not reveal any differences between control and treated groups which
could be related to treatment. Histopathological examination of pups
of generation F3 revealed no treatment-related effects. Survival,
growth and development of pups in treated groups were similar to those
of the control group apart from a slight delay in tooth eruption in
the 150 and 500 mg/kg groups of generations F1b and F2. No delay in
tooth eruption was seen in any of the treated groups of generation
F3.
In the teratological investigations, foetuses of all treated
groups in the generation F3 were slightly more advanced in their
degree of skeletal ossification compared to the control groups.
Finally, post mortem examinations of the dams of generations F0a, F1a
and F2 used in the teratology studies revealed no significant
differences between control and treated animals, except for slightly
increased numbers of corpora lutea and post-implantation losses in the
150 mg/kg group of generation F1a. These were considered to be
unrelated to treatment (Grant et al., 1979).
Acute toxicity
No information available.
Short-term studies
Mouse
Mice (number not stated) were given intraperitoneal injections of
a 1 to 2% aqueous solution of the lithium salt of carminic acid for a
period of 60 days. The only abnormality observed was proliferation of
spleen tissue (Harada, 1931).
Rat
Groups of 40 rats, equally divided by sex, received ammoniacal
cochineal carmine in 0.4% aqueous agar by intubation at dosage levels
of 0, 2.5, 5.0 and 10.0 g/kg 5 days per week for 13 weeks. Body weight
was recorded bi-weekly. Blood counts were made 3 times. Gross and
microscopic findings were not remarkable, aside from a dose-related
accumulation of colour in the tissues of the rats receiving the 2
higher dosage levels. No haematological effects were noted. At the 2
highest levels some decreased growth was apparent. Urine and faeces of
the treated rats were coloured during the period of administration
(Battelle, 1962).
Groups of 50 weanling rats equally divided by sex were fed
calcium carmine in the diet at levels of 0, 50, 250 and 500 mg/kg bw
per day for 90 days. Blood counts, blood glucose, blood urea nitrogen
and urinalyses were performed 3 times. No effects due to the carmine
were reported in terms of growth, haematology and other clinical
findings. Gross and microscopic pathology were not remarkable (FDRL,
1962).
Rabbit
Five rabbits were given intravenous injections every 5 to 7 days,
of 3 to 10 ml of a 2 to 4% aqueous solution of the lithium salt of
carminic acid. The treatment was continued for periods varying from
130 to 529 days. No tumours were observed, but great proliferation of
the tissue of the spleen was noted (Harada, 1931).
Long-term studies
Rat
Carmine was mixed with the diet of groups of 66 male and 66
female rats to provide daily intakes of 50, 150 or 500 mg/kg bw per
day for 8 weeks. A group of 114 male and 114 female rats fed the basic
diet served as controls. The treatment continued whilst each female
was mated with a male from the same group, during pregnancy and the
rearing of the resulting offspring. Young from these litters were used
to provide groups of 54 males and 54 females given the same dose
levels of carmine or 90 males and 90 females in a control group. The
treatment for each animal was the same as that of its parents and
continued until any one of the groups reached approximately 20%
survival. This resulted in the surviving males being killed in week
108 and the females in week 109. Feeding diets to provide intakes of
up to 500 mg/kg bw per day did not adversely affect the survival of
the rats, their growth or intakes of food and water.
Samples of blood taken at 3, 6, 12 and 18 months from some of the
animals and from all survivors showed no changes which could be
associated with treatment. Similarly, no treatment-related changes
were found in renal concentration tests and semi-quantitative analysis
of urine at 3, 6, 9, 12 and 18 months or in serum chemistry and organ
weights at the end of the study.
The incidence of tumours was not affected by treatment and most
of the non-tumour pathology could not be associated with treatment.
There were more females with mammary gland acinar hyperplasia and duct
ectasia in all treated groups than in controls and more with mild
changes of the stomach at the 2 higher doses. There was a small
increase in the incidence of animals with vaginitis or lymph node
oedema affecting the high dose females and distended hepatic sinusoids
in the high dose males. None of these findings were considered to be
due to treatment. It is concluded that carmine administered to rats
in utero and to 20% survival is not carcinogenic and that the no-
untoward-effect level is 500 mg/kg bw per day (Ford et al., 1981).
Comments
Several mutagenic studies using microorganisms both in vitro
and in host-mediated assay provide no evidence for potential
genotoxicity for carminic acid.
A teratogenicity study in which ammonium carmine was administered
by gastric intubation to pregnant rats did not show the findings of
earlier studies in which sodium or lithium carmines were injected i.p.
in pregnant mice. There were no significant effects on reproduction,
embryotoxicity, teratology or development when ammonium carmine was
administered in the diet to rats over 3 generations. Furthermore, a
long-term study in rats with ammonium carmine using animals exposed
in utero indicated that this compound is not carcinogenic.
The results of a short-term study in rats fed ammonium carmine
for 90 days at doses up to 10 g/kg failed to show toxic effects.
EVALUATION
Level causing no toxicological effect
Rat: 500 mg/kg bw in the diet.
Estimate of acceptable daily intake for man
0-5 mg/kg bw.
The ADI includes ammonium carmine or the equivalent of calcium,
potassium or sodium salts.
REFERENCES
Battelle Memorial Institute (1962) Unpublished report submitted to
WHO
Barale, R. et al. (1978) Evaluation of potential mutagenic activity
of carminic acid. In: Galli, C. L., Paoletti, R. & Vettorazzi,
G., eds, Proceedings of the International Symposium on Chemical
Toxicity of Food, Elsevier, North Holland Biomedical Press
Brown, J. P. & Brown, R. J. (1976) Mutagenesis by 9,10-anthraquinone
derivatives and related compounds in Salmonella typhimurium,
Mutation Res., 40, 203-224
Brown, J. P., Roehm, G. W. & Brown, R. J. (1977) Mutagenicity testing
of certified food colours and related azo, xanthene and
triphenylmethane dyes with Salmonella/microsome system,
Environ. Mutagen Soc. 8th Ann. Meet., Abst. p. 33
Food and Drug Research Laboratories (1962) Unpublished report
submitted to WHO
Ford, G. P. et al. (1981) Unpublished report from B.I.B.R.A.,
submitted to WHO. Long-term study in rats with carmine of the
cochineal using animals exposed in utero, Report, 230/1/81
Gaunt, I. F., Clode, S. A. & Lloyd, A. G. (1976) Unpublished report
from B.I.B.R.A., submitted to WHO. Studies of teratogenicity and
embryotoxicity of carmine in the rat, Report, 162/1/76, July 1976
Grant, D., Conning, D. M. & Hawkins, R. I. (1979) Unpublished report
from B.I.B.R.A, submitted to WHO. Multigeneration toxicity
studies in rats with carmine of cochineal, Report, 230/1/79,
December 1979
Harada, M. (1931) cited by Hartwell, J. L.: Survey of compounds which
have been tested for carcinogenic activity, 2nd ed., 1951, p. 118
Haveland-Smith, R. B. & Combes, R. D. (1980) Screening of food dyes
for genotoxic activity, Foods Cosmet, Toxicol., 18, 215-221
Kada, T., Tutikawa, K. & Sadaie, Y. (1972) In vitro and
host-mediated rec-assay procedures for screening chemical
mutagens and phloxine, a mutagenic red dye detected, Mutation
Res., 16, 165-174
Lloyd, A. G. (1980) Extraction and chemistry of cochineal,
Food Chem., 5, 91-107
Meloan, S. N., Valentine, L. S. & Puchtler, H. (1971) Histochemie,
27, 87
Sarkany, R. H., Meara, R. H. & Everall, J. (1961) Cheilitis due to
carmine in lip salve, Trans. St. John's Hosp. derm. Soc.
(Lond.), 48, 30-40
Schluter, G. (1970) Uber die embryotoxische Wirkung von Carmin bei der
Maus (Embryotoxic action of carmine in mice), Z. Anat.
Entwickl.-Gesch., 131, 228-235
Schluter, G. (1971a) Effects of lithium carmine and lithium carbonate
on the pre-natal development of mice, Naunyn-Schmiedebergs
Arch. Pharmak., 270, 56-64
Schluter, G. (1971b) Time-response relationship of embryotoxic effects
of lithium carmine in mice, Naunyn-Schmiedebergs Arch.
Pharmak., 270, 316-318
See Also:
Toxicological Abbreviations
CARMINES (JECFA Evaluation)