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    FAST GREEN FCF

    EXPLANATION

         Fast Green FCF was evaluated at the twenty-fifth meeting of the
    Committee (Annex 1, reference 56) when inadequacies were identified in
    earlier long-term feeding studies in rats and mice. The previously-
    allocated ADI was converted to a temporary ADI of 12.5 mg/kg b.w.
    pending the results of adequate long-term feeding studies and
    multigeneration reproduction/teratogenicity studies.

         Since the previous evaluation, additional data have become
    available and are summarised and discussed in the following monograph.
    The previously-published monograph has been expanded and is reproduced
    in its entirety below.

    BIOLOGICAL DATA

    Biochemical aspects

         Rats and dogs were given orally 200 mg of the colour. In the rats
    the urine and faeces were collected for 36 hours. In the dogs, a bile
    fistula was made for bile analysis. Almost all the administered colour
    was excreted unchanged in the faeces of rats. No colour was found in
    the urine. In the bile of the dogs, the amount of colour never
    exceeded 5% of the given dose. After feeding, the colour was found in
    the bile of rats and rabbits, but not in their urine. It was concluded
    that the quantity found in the bile provides a reasonable estimate of
    the amount absorbed from the gastrointestinal tract (Hess & Fitzhugh,
    1953; 1954; 1955).

         Following i.v. injection in rats, over 90% of the colour was
    excreted in the bile within 4 hours (Iga et al., 1971).

         Fast Green FCF was found to have a high binding affinity for
    plasma protein (Gangolli et al., 1967; 1972; Iga et al., 1971).

    Toxicological studies

    Special studies on carcinogenicity (see also long-term studies)

    Mice

         Groups of 60 (120 controls) male and female Charles-River CD-1
    mice were fed diets containing 0, 0.5, 1.5, or 5.0% Fast Green FCF
    from 43 days of age for approximately 24 months. Ten animals/sex/group
    were subjected to haematological examination at 3, 6, 12, and 18
    months. All animals dying or killed in a moribund condition and all
    survivors to termination were subjected to detailed post-mortem

    examination. The following tissues were examined histologically from
    all survivors from the control and 5%-dose groups as well as all
    animals dying or killed in extremis from these groups: adrenals,
    aorta, bone and marrow (femur), brain (3 sections), eyes (with optic
    nerve), gall bladder, gastrointestinal tract (oesophagus, stomach,
    duodenum, ileum, caecum, colon), heart, kidneys, liver, lung, lymph
    nodes (mesenteric and mediastinal), mammary gland, nerve (sciatic),
    ovaries, pancreas, pituitary, prostate, salivary gland, seminal
    vesicles, skeletal muscle, skin, spinal cord, spleen, testes with
    epididymides, thymus, thyroid/parathyroid, trachea, urinary bladder,
    uterus, and gross lesions/tissue masses. In addition, gross
    changes/tissue masses were examined histologically from all animals in
    the lower-dose groups.

         No treatment-related effects on mortality were observed. The mean
    body weights of females in the 5%-dose group were consistently lower
    than controls and the mean body weights of males in the 5%-dose group
    were lower than controls at weeks 52 and 78. No other consistent
    differences in body weight were noted. Slight reductions in
    haemoglobin, haematocrit, and erythrocyte counts were noted in the
    high-dose males at 18 months but no other consistent or dose-related
    haematological changes were observed. Histological examination did not
    reveal any treatment-related lesions and the incidence, origins,
    and histology of benign and malignant neoplasms did not differ
    significantly between controls and treated animals (Hogan & Knezevich,
    1981).

    Rats

         Eighteen weanling Osborne-Mendel rats of both sexes received
    weekly s.c. injections of approximately 30 mg (1 ml of a 3% aqueous
    solution) of Fast Green FCF for 94-99 weeks. Subcutaneous
    fibrosarcomas appeared at the site of injection in 15 animals (Nelson
    & Hagan, 1953; Hansen et al., 1966).

         Two groups of 16 female rats (control groups of 10 rats) were
    given s.c. injections of 0.5 ml of a 3% or 6% solution (the rats
    received with each injection 15 or 30 mg, respectively). The colour
    used in the experiment was certified as 92% pure and was supplied as
    the disodium sulfonate salt. The 10 control rats were given distilled
    water injections. At first, injections of 6% were given 3 times a
    week; after 17 weeks it became necessary to reduce the dose to 3%.
    Thereafter, both groups were given injections of 3% twice weekly for
    9 weeks. The rest of the time, 22 weeks, both groups were injected
    usually once a week, while occasionally 2 injections were tolerated.
    Growth inhibition was observed. Thirteen out of 16 animals receiving
    6% of the colour had fibrosarcomas. The animals given 3% also showed
    fibrosarcomas (10 out of 12). The controls did not show neoplastic
    tissue at the site of injection (Hasselbach & O'Gara, 1960).

         Subcutaneous injection of 1 ml of an 0.8% solution twice weekly
    produced histological changes suggestive of subsequent sarcoma
    formation unassociated with chemical carcinogenic potential (Grasso &
    Golberg, 1966).

         No tumours were produced in 11 hamsters injected with 1 mg of the
    dye in 0.1 ml water (Price et al., 1978).

         A carcinogenicity study with an in utero phase was carried out
    in Charles-River albino rats. Groups of 60 (120 controls) male and
    female rats of the F0 generation were fed diets containing 0, 1.25,
    2.5, or 5.0% Fast Green FCF for 2 months prior to mating and
    throughout gestation and lactation. Following the reproductive phase,
    a maximum of 4 animals of each sex/litter were randomly selected from
    the F1 generation for the long-term carcinogenicity study. Groups of
    70 animals of each sex/group were given Fast Green FCF in the diet at
    the same concentrations as the parent generation. An interim kill of
    10 animals of each sex per group was carried out after 12 months; the
    remaining animals continued to receive the test diets for 29 months
    (males) or 31 months (females). Haematology, clinical chemistry tests,
    and urinalysis were performed on 10 rats of each sex/group at 3, 6,
    12, 18, and 24 months. Gross autopsies were performed on all animals
    that died on test or were killed in a moribund condition and on all
    F1 generation animals at interim and terminal sacrifice. The
    following tissues were examined histologically from all animals killed
    at interim sacrifice and all survivors from the control and 5%-dose
    groups, as well as all animals dying or killed in extremis from
    these groups: adrenals, aorta, bone and marrow (femur), brain
    (3 sections), eyes (with optic nerve), gastrointestinal tract
    (oesophagus, stomach, duodenum, ileum, caecum, colon), heart, kidneys,
    liver, lung, lymph nodes (mesenteric, mediastinal) mammary gland,
    nerve (sciatic), ovaries, pancreas, pituitary, prostate, salivary
    gland, seminal vesicles, skeletal muscle, skin, spinal cord, spleen,
    testes with epididymides, thymus, thyroid/parathyroid, trachea,
    urinary bladder, uterus, and gross lesions/tissue masses. In addition,
    gross changes/tissue masses were examined histologically from all
    animals in the lower-dose groups. Subsequently, the urinary bladder
    from males of the 1.25- and 2.5%-dose groups were also examined
    histologically.

         During the premating period, no treatment-related effects were
    seen on mortality or body-weight gain but there was a dose-related
    increase in food consumption. After mating there were no treatment-
    related effects on the number of successful pregnancies or pup
    viability at birth, but pup mortality was increased in the 5%-dose
    group during the period 4-14 days of lactation. Mean pup weight was
    reduced in all treated groups, most markedly in the high-dose group.

         In the F1 generation, mortality was slightly higher in all
    treated groups than in controls, but it did not vary in a dose-related
    manner. Mean body weights of the high-dose males were consistently
    lower than controls, even though their food intake was elevated.
    Fasting blood glucose levels were elevated in females in all treated
    groups at 3 and 12 months, females in the 1.25 and 2.5% groups at 18
    months, and males in all treated groups at 12 and 18 months.

         At interim (12 months) sacrifice, the mean absolute and relative
    thyroid weights were elevated in the high-dose males while the
    relative kidney weights were elevated in the high-dose females. At
    termination, the thyroid weights were elevated in males of the
    2.5- and 5%-dose groups and females of the 5% group; kidney weights
    were elevated in both sexes of the 5%-dose group and females of the
    2.5% group. No treatment-related effects were seen in urinalyses,
    haematology determinations, physical observations, or ophthalmology.

         Histopathological examination revealed an increased incidence of
    urothelial hyperplasia in treated males and of urinary bladder
    transitional cell/urothelial neoplasms in males of the 5%-dose group.
    The overall incidences in males are summarized below:

                                                                        

    Group                      Control    Control   1.25%    2.5%    5%
                               1          2
                                                                        

    Number examined            58         61        58       55      60

    Number with neoplasia      1          2         1        2       5

    Number with hyperplasia    1          4         7        10      3
                                                                        

         Non-statistically-significant increases in testicular Leydig cell
    tumours and neoplastic nodules in the liver were also observed. When
    time-to-tumour analysis was performed on pathology incidence data, the
    increased incidence of bladder tumours was confirmed and the incidence
    of several other tumour types showed statistically-significant
    differences related to treatment, including neoplastic nodules in the
    liver (males and females), female mammary adenomas and pituitary
    adenomas, male parthyroid adenomas, male thyroid medullary carcinomas,
    female uterine leiomyosarcomas, and male testicular
    interstitial/Leydig cell tumours.

         Of the non-neoplastic pathology, chronic nephropathy was a common
    finding in all groups but the severity was greater in females in the
    5%-dose group. Other lesions did not appear to be related to treatment
    (Knezevich & Hogan, 1981).

    Special studies mutagenicity

         Fast Green FCF was non-mutagenic in the Salmonella/microsome
    assay (Brown et al., 1978) and negative results were also obtained
    in bacterial DNA repair tests (Kada et al., 1972; Rosenkranz &
    Leifer, 1980). The colour was inactive in a gene conversion assay in
    diploid yeast (Sankaranarayanan & Murthy, 1979).

         In one of 2 experiments, colour-induced cell transformation
    occurred in cultured Fisher rat embryo cells at a concentration of
    1 g/ml (Price et al., 1978) and chromosome damage was reported in
    an in vitro test using Chinese hamster ovary cells (Au & Hsu, 1979).

    Special studies on reproduction

    Rats

         A 3-generation reproduction study was carried out on Fast Green
    FCF in Long-Evans rats at dose levels of 0, 10, 100, 300, or
    1,000 mg/kg b.w./day. The first generation parents (10 males, 20
    females) were given the appropriate dose of Fast Green FCF in the diet
    2 weeks before the first mating, and dosing continued throughout the
    gestation, lactation, and post-weaning phases for three successive
    generations. The F0 generation rats were mated twice, the F1a
    litters being necropsied at weaning, and selected animals (10 males,
    20 females) from the F1b litters were used for breeding.

         Following an 80-day growth period, animals from the F1b
    generation were mated 3 times and the offspring of the F2a and F2b
    generations were treated identically to the F1a and F1b generations.
    Following the third mating, half of the pregnant dams were sacrificed
    on day 19 of gestation, the uterine contents were examined for total
    embryos/resorption sites, and the corpora lutea per ovary were
    recorded. The other half were allowed to deliver normally (F2c) and
    were sacrificed at weaning.

         The F2b animals were mated once and allowed to raise their
    offspring to weaning when both parents and offspring were culled.

         Gross necropsies were performed on all parent animals and on
    F1a, F2a, F2c, and F3a offspring at weaning. Selected tissues from
    5 animals of each sex/dose from the F1b parents and the F3a
    generation at weaning were fixed at necropsy, and the following
    tissues examined histologically from the control and high-dose group:
    stomach, ileum, jejunum, colon, liver, spleen, heart, lungs, adrenals,
    kidneys, urinary bladder, thyroid, ovaries, and uterus or testes.

         No effects attributable to treatment were observed with respect
    to food consumption, body weight, adult mortality, mating performance,
    pregnancy and fertility rates, gestation length, offspring survival,
    weights and sex, litter survival, resorption rates, or necropsy
    findings. There were no macroscopic or microscopic tissue
    abnormalities of either F1b- or F3a-generation animals considered to
    be attributable to treatment (Smith, 1973).

    Acute toxicity
                                                             

                           LD50
    Species     Route      (mg/kg b.w.)      Reference
                                                             

    Rat         Oral       > 2,000           Lu & Lavallee,
                                             1964

    Dog         Oral       > 200             Radomski &
                           mg/dog            Deichman, 1956
                                                             

    Short-term studies

    Dogs

         Four beagles/group, equally divided by sex, were fed Fast Green
    FCF at 0, 1.0, or 2.0% of the diet for 2 years. Histopathology
    attributable to the colour was limited to green blobs of pigment in
    the renal cortical tubular epithelial cytoplasm of a male dog at the
    high-dose level; a female dog at the high-dose level showed slight
    interstitial nephritis and slight bone marrow hyperplasia (Hansen
    et al., 1966).

    Long-term studies

    Mice

         Groups of 50 male and 50 female C3HeB/FeJ mice were fed diets
    containing 1.0 or 2.0% Fast Green FCF for 2 years and 100 mice of each
    sex served as controls. After 78 weeks, 56 controls, 27 animals in the
    1.0%-treatment group, and 17 animals in the 2.0%-treatment group still
    survived. Microscopic examination revealed no lesions that were
    attributed to feeding of the colour (Hansen et al., 1966).

    Rats

         Groups of 50 weanling Osborne-Mendel rats, evenly divided by sex,
    were fed diets containing 0, 0.5, 1.0, 2.0, or 5.0% colour for 2
    years. No effects on growth or mortality were observed. Microscopic
    examination revealed no lesions that were attributed to the feeding of
    the colour (Hansen et al., 1966).

         The colour was fed at a dietary level of 4.0% to 5 male and 5
    female rats for periods from 18 to 20 months. This procedure resulted
    in gross staining of the forestomach, glandular stomach, small
    intestine, and colon. Granular deposits were noted in the stomach. No
    tumours were observed (Willheim & Ivy, 1953).

    Observations in man

         No data available.

    Comments

         The production of local sarcomata at the site of s.c. injection
    in rats is not considered to constitute evidence of carcinogenicity by
    the oral route. The mouse oral carcinogenicity study was negative but
    in the rat study, an increased incidence of urothelial hyperplasia
    and/or neoplasia of the bladder was observed. The biological
    significance of observed differences in benign and malignant tumours
    at other sites is questionable since, in some cases, statistically-
    significant differences were observed between the 2 control groups
    and, apart from the bladder, complete histological examination was not
    performed on the low- and intermediate-dose groups.

         Biochemical studies have shown that the colour is poorly absorbed
    and the 3-generation reproduction/teratogenicity study was uneventful.

         In view of the equivocal results of the most recent
    carcinogenicity study in rats, the evaluation is based on the earlier
    study, pending complete histological examination of all groups of rats
    and biometric examination of the data.

    EVALUATION

    Level causing no toxicological effect

    Mouse:    5% in the diet equal to 18,600 mg/kg b.w./day falling to
              8,000 mg/kg b.w./day.

    Rat:      5% in the diet equivalent to 2,500 mg/kg b.w./day.

    Estimate of temporary acceptable daily intake for man

    0-12.5 mg/kg b.w.

    Further work or information

    Required by 1986

         Complete histological examination of all dose-groups in the long-
    term carcinogenicity feeding-study in the rat and biometric
    examination of the data.

    REFERENCES

    Au, W. & Hsu, T.C. (1979). Studies on clastogenic effects of biologic
         stains and dyes, Environmental Mutagenesis, 1, 27.

    Brown, J.P., Roehm, G.W., & Brown, R.J. (1978). Mutagenicity testing
         of certified food colours and related azo, xanthene and
         triphenylmethane dyes with the Salmonella/microsome system,
         Mutation Res., 56, 249-271.

    Gangolli, S.D., Grasso, P., & Golberg, L. (1967). Physical factors
         determining the early local tissue reactions produced by food 
         colourings  and  other  compounds  injected subcutaneously.
         Fd. Cosmet. Toxicology., 5, 601-621.

    Gangolli, S.D., Grasso, P., Golberg, L., & Hooson, J. (1972). Protein
         binding by food colourings in relation to the production of
         subcutaneous sarcoma.  Fd. Cosmet. Toxicology., 10, 449-462.

    Grasso, P. & Golberg, L. (1966). Subcutaneous sarcoma as an index
         of carcinogenic potency. Fd. Cosmet. Toxicology., 4, 297-320.

    Hansen, W.H., Long, E.L., Davis, K.J., Nelson, A.A., & Fitzhugh,
         O.G. (1966). Chronic toxicity of three food colourings: guinea
         green B, light green SF yellowish, and fast green FCF in rats,
         dogs and mice, Fd. Cosmet. Toxicology., 4, 389-410.

    Hess, S.M. & Fitzhugh, O.G. (1953). Metabolism of coal-tar dyes.
         I. Triphenylmethane dyes. Fed. Proc., 12, 330-331.

    Hess, S.M. & Fitzhugh, O.G. (1954). Metabolism of coal-tar dyes.
         II. Bile studies. Fed. Proc., 13, 365.

    Hess, S.M. & Fitzhugh, O.G. (1955). Absorption and excretion of
         certain triphenylmethane colours in rats and dogs,
         J. Pharmacol. Exp. Ther., 114, 38-42.

    Hesselbach, M.L. & O'Gara, R.W. (1960). Fast green and light green
         induced tumours: induction, morphology and effect on host.
         J. Nat. Cancer Inst., 24, 769-793.

    Hogan, G.K. & Knezevich, A.L. (1981).  A long-term oral
         carcinogenicity study of FD&C Green No. 3 in mice. Unpublished
         report No. 77-1781 from Bio/dynamics Inc., East Millsone, NJ,
         USA. Submitted to WHO by Certified Color Manufacturers'
         Association.

    Iga, T., Awazu, S., & Nogami, H. (1971). Pharmacokinetic study of
         biliary excretion. II. Comparison of excretion behaviour in
         triphenylmethane dyes. Chem. Pharm. Bull., 19, 273-281.

    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.

    Knezevich, A.L. & Hogan, G.K. (1981). A long-term oral toxicity/
         carcinogenicity study of FD&C Green No. 3 in rats. Unpublished
         report No. 77-1780 from Bio/dynamics Inc., East Millstone, NJ,
         USA. Submitted to WHO by Certified Color Manufacturers'
         Association.

    Lu, F.C. & Lavallee, A. (1964). The acute toxicity of some synthetic
         colours used in drugs and food. Canad. Pharm. J., 97, 30.

    Nelson, A.A. & Hagan, E.C. (1953). Production of fibrosarcomas in rats
         at site of subcutaneous injection of various food dyes. Fed.
         Proc., 12, 397-398.

    Price, P.J., Suk, W.A., Freeman, A.E., Lane, W.T., Peters, R.L.,
         Vernon, M.L., & Huebner, R.J. (1978). In vitro and in
         vivo indications of the carcinogenicity and toxicity of food
         dyes. Int. J. Cancer, 21, 361-367.

    Radomski, J.L. & Deichman, W.B. (1956). Cathartic action and
         metabolism of certain coal tar food dyes. J. Pharmacol. Exp.
         Ther., 118, 322-327.

    Rosenkranz, H.S. & Leifer, Z. (1980). In Chemical Mutagens:
         Principles and Methods for their Detection. Ed: de Serres,
         F.J., & Hollaender, A. Plenum Press; New York & London, Vol. 6,
         p. 109.

    Sankaranarayanan, M. & Murthy, M.S.S. (1979). Testing of some
         permitted food colours for the induction of gene conversion in
         diploid yeast. Mutation Res., 67, 309-314.

    Smith, J.M. (1973). A three generation reproduction study of FD&C
         Green No. 3 in rats. Unpublished report No. 71R-736 from
         Bio/dynamics Inc., East Millstone, NJ, USA. Submitted to WHO by
         Certified Color Manufacturers' Association.

    Willheim, R. & Ivy, A.C. (1953). A preliminary study concerning the
         possibility of dietary carcinogenesis. Gastroenterology, 23,
         1-19.
    


    See Also:
       Toxicological Abbreviations
       Fast green FCF  (FAO Nutrition Meetings Report Series 46a)
       Fast Green FCF (WHO Food Additives Series 16)
       Fast Green FCF (WHO Food Additives Series 21)
       FAST GREEN FCF (JECFA Evaluation)
       Fast Green FCF (IARC Summary & Evaluation, Volume 16, 1978)