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.,

         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).



         No information on metabolism is available.


    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

         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

         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


         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).


         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,


         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


         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).


         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.


    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.


    Battelle Memorial Institute (1962) Unpublished report submitted to

    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)