CARMINES (FORMERLY COCHINEAL, CARMINE, AND CARMINIC ACID)
Explanation
Cochineal, carmines and carminic acid were evaluated for
acceptable daily intake for man by the Joint FAO/WHO Expert Committee
on Food Additives in 1975 and 1977 (see Refs. 37 and 43 in the Annex).
Since the previous evaluations, additional data have become
available and the previous monographs have 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 et al., 1977).
Carminic acid did not produce reverse mutations in four 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 assays 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.
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/kg bw of carmine on days 6, 8, 10, 12 or 14 of
pregnancy. A teratogenic effect was observed on the first three
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 numbers
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 three 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 FoA) 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 FoA, 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 bw 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 FoA, 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 bw 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 bw five days per week for 13 weeks.
Body weight was recorded bi-weekly. Blood counts were made three
times. Gross and microscopic findings were not remarkable aside from a
dose-related accumulation of colour in the tissues of the rats
receiving the two higher dosage levels. No haematological effects were
noted. At the two 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/day for 90 days. Blood counts, blood glucose, blood urea nitrogen
and urinalyses were performed three 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 five to
seven 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).
Comments
Cochineal and colouring principles derived from it were
considered in the twenty-first report of the Expert Committee but at
that time the toxicological data available were considered to be
insufficient for an evaluation and the establishment of an ADI. The
present Committee understood that the main substances used were
ammonium carmine for alcoholic beverages and calcium carmine for
foods. Their use was limited because of the small amount of cochineal
produced. Recent reproduction studies with ammonium carmine had found
no toxicologically significant effects. The Committee was informed
that a long-term study had also been completed, but the data had not
yet been submitted. The Committee allocated a temporary ADI of
0-2.5 mg/kg bw for ammonium carmine, or equivalent amounts of the
calcium, potassium, or sodium salts (the lithium salt is not
acceptable for food-additive use). This was based on a no-effect
level of 500 mg of ammonium carmine per kg of body weight in a
multigeneration study. The Committee requested the submission of the
results of the long-term study for evaluation at a future meeting.
EVALUATION
Level causing no toxicological effect
Rat: 500 mg/kg bw per day.
Estimate of temporary acceptable daily intake for man
0-2.5 mg/kg bw.
FURTHER WORK OR INFORMATION
Required by 1982.
Submission of the results of the long-term studies.
REFERENCES
Battelle Memorial Institute (1962) Unpublished report submitted to WHO
Barale, R. et al. (1978) In: Galli, C. L., Paoletti, R. & Vettorazzi,
G., eds, Proceedings of the International Symposium on Chemical
Toxicology of Food, Elsevier, North Holland Biomedical Press,
pp. 349-357
Brown, J.P. & Brown, R. J. (1976) Mutation Res., 40, 203
Brown, J. P., Roehm, G. W. & Brown, R. J. (1977) Environ, Mutagen
Soc. 8th Ann. Meet., Abst., p. 33
Food and Drug Research Laboratories (1962) Unpublished report
submitted to WHO
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
Haveland-Smith, R. B. & Combes, R. D. (1980) Foods and Cosmetics
Toxicology, 18, 215-221
Lloyd, A. G. (1980) Food Chemistry, 5, 91-107
Meloan, S. N., Valentine, L. S. & Puchtler, H. (1971) Histochemie,
27, 87
Kada, T., Tutikawa, K. & Sadaie, Y. (1972) Mutation Res., 16, 165
Sarkany, R. H., Meara, R. H. & Everall, J. (1961) Trans. St. John's
Hosp. derm. Soc. (Lond.), 48, 39
Schluter, G. (1970) Z. Anat. Entwickl.-Gesch., 131, 228
Schluter, G. (1971a) Naunyn-Schmiedeberg's Archiv. Pharmak., 270,
56
Schluter, G. (1971b) Naunyn-Schmiedeberg's Archiv. Pharmak., 270,
316