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    CANTHAXANTHIN

    First draft prepared by
    Dr Preben Olsen
    Institute of Toxicology, National Food Agency
    Ministry of Health, Soborg, Denmark

    Explanation
    Biological data
         Biochemical aspects
         Absorption, distribution, and excretion
         Effect on enzymes and other biochemical
           parameters
    Toxicological studies
         Long-term toxicity/carcinogenicity studies
         Special studies on ocular toxicity
         Special studies on immune responses
         Observations in humans
    Comments
    Evaluation
    References

    1.  EXPLANATION

         Canthaxanthin was previously evaluated at the tenth,
    eighteenth, thirty-first and thirty-fifth meetings of the Committee
    (Annex 1, references 13, 35, 77 and 88). At the thirty-first
    meeting, the Committee noted that canthaxanthin had been used as a
    direct food additive, as a feed additive, and as an orally
    administered pigmenting agent for human skin in both pharmaceutical
    and cosmetic applications. The previous ADI was reduced to
    0-0.05 mg/kg bw and made temporary pending submission of (1)
    details of ongoing long-term studies in rats and mice; (2)
    clarification of the factors that influence pigment deposition in
    the eye, including the establishment of the threshold dose, the
    influence of dose and duration of exposure, the reversibility of
    pigment accumulation, and the investigation of potential animal
    models; and (3) clarification of whether pigment deposition is
    causally related to impaired ocular function. At the thirty-fifth
    meeting, the Committee concluded that the long-term toxicity of
    canthaxanthin in rats indicated potential hepatotoxicity in humans.
    However, it considered that the main problem associated with
    canthaxanthin was the deposition of crystals in the human retina.
    In view of the irreversibility or very slow reversibility of such
    retinal crystal deposition, the significance of which was not
    known, the Committee was unable to establish an ADI for
    canthaxanthin when used as a food additive or animal feed additive.
    The previous temporary ADI was therefore not extended.

         Since the previous evaluation, additional data have become
    available and are summarized and discussed in the following
    monograph addendum.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

         Single radiolabelled doses of 0.2 or 0.6 mg/kg bw of
    14C-canthaxanthin were administered to male and female Cynomolgus
    monkeys. Blood and plasma profiles were similar in males and
    females. Faecal excretion was the major route of elimination of the
    radiolabelled dose (84-89%), urinary excretion accounted for
    1.6%-3.6%, and 1.6%-4.6% was retained in tissues. About 3%-7% of
    the dose was absorbed. Of the amount absorbed, the highest
    concentrations were found in the adrenal gland (3.2-8.6 g
    equivalent 14C-canthaxanthin/g at the high dose), with moderate
    levels in the spleen, liver, bone marrow, skin, and fat (0.1-0.9 g
    equivalent 14C-canthaxanthin/g at the high dose). Low levels of
    radioactivity were found in parts of the eye and brain at the high
    dose (0.01-0.05 g equivalent 14C-canthaxanthin/g) (Bausch, 1992a).

         Canthaxanthin metabolism was compared in rats and monkeys
    using radiolabelled 14C-canthaxanthin administered orally at dose
    levels of 0.2 or 0.6 mg/kg bw to each animal species. Canthaxanthin
    was metabolized and excreted faster in rats than in monkeys. The
    concentrations of radioactivity in rat tissues were less than 1%
    after 96 h, compared to 7.4% in monkey tissues. Compared to
    monkeys, the adrenals were not a target organ for retention of
    radioactivity in rats. In both species, noticeably low levels were
    found in the eye with about 100-fold lower concentrations in the
    rat (Bausch, 1992b).

         In order to determine whether canthaxanthin accumulation in
    the eye was dependent on the presence of melanin, the accumulation
    of canthaxanthin in pigmented rats was investigated and compared to
    data obtained in albino rats. Male pigmented PGV/LacIbm and male
    albino rats (strain not specified) were given canthaxanthin at a
    dietary level of 100 mg/kg of feed for 5 weeks. At termination, the
    tissue concentration of canthaxanthin in pigmented rats compared to
    albino rats was more than 10 times lower in spleen, liver and skin,
    about 2 times lower in small intestine and kidney fat, and 6
    times lower in eyes (canthaxanthin concentrations in the eyes
    of pigmented and albino rats were 0.02 g/g and 0.13 g/g,
    respectively). The authors concluded that the pigmented rat was not
    a better model for canthaxanthin deposits than the albino rat
    (Bausch  et al., 1991).

         The distribution of the radioactivity of 6,7,6',7'-14C-
    canthaxanthin was studied in male rats, receiving 0, 0.001 or 0.01%
    unlabelled canthaxanthin in the diet for 5 weeks in order to
    achieve steady state conditions. A single dose of radiolabelled
    canthaxanthin was given either as a 2 ml liposomal preparation into
    the stomach or in beadlets mixed in diet The pattern of
    distribution in the tissues (liver, spleen, heart, lungs, thymus,
    kidneys, adrenal glands, testes, epididymis, eyes, brain, skin,
    stomach, small and large intestines) and of faecal and urinary
    excretion was found to be similar for all preparations and
    applications. After 1 day, 46-89% of the applied radioactivity was
    excreted, and more than 98% was excreted after 7 days (Glatzle &
    Bausch, 1989).

    2.1.2  Effect on enzymes and other biochemical parameters

         Canthaxanthin inhibited, in a dose-related manner, the
     in vitro prostaglandin biosynthesis by squamous carcinoma cells
    in culture (El-Attar & Lin, 1991).

         Liver content of cytochrome P-450, and the activity of
    NADH-cytochrome c reductase, and some P-450 dependent enzymes were
    increased in male rats given canthaxanthin at a dietary level of
    300 mg/kg of diet indicating that canthaxanthin was an inducer of
    liver xenobiotic-metabolizing enzymes (Astorg  et al., 1994).

    2.2  Toxicological studies

    2.2.1  Long-term toxicity/carcinogenicity studies

    2.2.1.1  Mice

         Dietary concentration of 1% canthaxanthin resulted in a 50%
    reduction in primary UV-induced skin tumours (expressed as affected
    skin per unit area) in mice compared to controls fed a basal diet.
    Dietary supplementation with a combination of canthaxanthin and
    retinyl palmitate resulted in further reduction of tumour incidence
    (Rybski  et al., 1991) and prevented the transfer of ultraviolet-
    induced immunosuppression with splenocytes from ultraviolet type B
    irradiated mice (Gensler, 1989).

    2.2.1.2  Rats

         Groups of 50 male CD Sprague-Dawley rats were given
    canthaxanthin incorporated in the diet at doses of 0, 0 (placebo),
    5, 25, 75 or 250 mg/kg bw/day for up to 104 weeks. The
    canthaxanthin was micro-encapsulated in water soluble beadlets
    containing 10% canthaxanthin. Similar beadlets devoid of
    canthaxanthin (placebo beadlets) were also prepared. The test
    animals received a constant dietary concentration of beadlets, the

    different dose levels being achieved by mixing appropriate
    proportions of canthaxanthin and placebo beadlets. One control
    group received regular diet and the second (placebo) control group
    received a similar concentration of placebo beadlets as was given
    to the test animals. The mean intakes of canthaxanthin were 99-100%
    of the target dose. In each group, 10 animals were assigned for
    interim sacrifice after 52 weeks and another 10 animals after 78
    weeks of treatment; 30 animals were treated for 104 weeks. Ten
    animals/group were also subjected to laboratory investigations
    (haematology, clinical chemistry, urinalysis) after 26, 51, 78 and
    104 weeks of treatment. Ophthalmoscopy was scheduled for all groups
    pre-dose and after 51 and 104 weeks of treatment. A detailed
    necropsy was performed on rats after spontaneous death or scheduled
    sacrifice. Histopathology was limited to the examination of the
    liver of all animals.

         No treatment-related effects were seen on survival of rats.
    Progressive red staining of the fur and tail were observed in a
    proportion of animals from the 25 mg/kg bw/day and higher dose
    groups. Mean body-weight gains of animals which received placebo
    and/or canthaxanthin formulation were generally inferior to the
    weight gains of untreated controls, but this trend was not
    statistically significant. A slight reduction of weight gain
    compared with the placebo control was seen at 25 mg/kg bw/day and
    higher doses during the first 17 weeks of the test. Food
    consumption was comparable in all groups throughout the treatment
    period. Eye examinations showed no abnormalities related to
    treatment after 51 and 104 weeks. Haematological parameters showed
    no intergroup differences attributable to treatment, whereas
    clinical chemistry changes were limited to a marginally higher mean
    plasma cholesterol level in animals treated with 250 mg/kg bw/day,
    and a slightly higher activity of alkaline phosphatase in animals
    treated with 75 and 250 mg/kg bw/day after 104 weeks. No intergroup
    differences were observed in urine parameters. There were no
    organ-weight changes at the interim and terminal sacrifices. Gross
    pathological examination at interim sacrifice and at termination
    revealed orange/red discolouration of the GI tract and orange
    discolouration of the subcutis and adipose tissue at all dose
    levels. Discolouration of the liver was seen at the high doses, in
    the 25 mg/kg bw/day dose group at week 78, and in a few animals
    from the 5 mg/kg bw/day dose group at termination of the test.

         Histopathological examination of animals at interim and
    terminal sacrifices revealed treatment-related increases in the
    incidence or severity of lesions in the liver. Hepatocyte
    enlargement was found in all animals receiving 75 and 250 mg/kg
    bw/day. Increased incidences of vacuolation were observed at
    25 mg/kg bw/day (at week 52 only), and at 75 and 250 mg/kg bw/day
    (at weeks 52 and 104) when compared to untreated control and
    placebo control. Ground glass cells were observed among animals

    treated with 75 and 250 mg/kg bw/day after 78 weeks. After 78
    weeks, a higher grade of periportal fat accumulation was noted in
    animals treated with 75 and 250 mg/kg bw/day, extending to a higher
    incidence and/or grade of generalized fat accumulation after 104
    weeks, when compared with the relatively high background of fatty
    change seen in both controls. Birefringent orange/brown pigment in
    hepatocytes was observed at dose levels of 75 and 250 mg/kg bw/day
    after 52 weeks, and at doses of 25 mg/kg bw/day and above after 78
    and 104 weeks. There was no evidence of an increased incidence of
    liver cell tumours in canthaxanthin-treated rats in comparison with
    controls. Two benign liver cell tumours were found in the 250 mg/kg
    bw/day group. One malignant liver cell tumours was found in each of
    the untreated control and 250 mg/kg bw/day dose group. It was
    concluded that oral treatment with 5 and 25 mg canthaxanthin/kg
    bw/day was not associated with liver impairment (Buser, 1992a).

         In a similar study, groups of 80 to 105 female CD Sprague
    Dawley rats were given canthaxanthin incorporated in the diet at
    dose levels of 0, 0 (placebo), 5, 25, 75 or 250 mg/kg bw/day. In
    each group, 10 animals were sacrificed after 52 weeks of treatment,
    another 10 animals after 78 weeks, and 60 animals were treated for
    104 weeks. In addition, a 26-week recovery period was scheduled for
    10 additional animals from groups receiving placebo, 75 and
    250 mg/kg bw/day canthaxanthin after 52 weeks of treatment, and for
    another 15 animals from the same groups including untreated control
    after 78 weeks of treatment. Ten animals/group were also subjected
    to laboratory investigations (haematology, clinical chemistry,
    urinalysis) after 26, 51, 78 and 104 weeks of treatment.
    Ophthalmoscopy was scheduled for all groups pre-dose and after 51
    and 104 weeks of treatment. A detailed necropsy was performed on
    all spontaneous deaths and scheduled sacrifices. Histopathology was
    limited to examination of the liver in all animals.

         No treatment-related adverse effects were seen on survival.
    Red staining of the fur and tail were observed in animals given
    25 mg/kg bw/day or higher doses; discolouration diminished in
    recovery animals withdrawn from treatment. Mean body-weight gain of
    animals receiving placebo or 250 mg/kg bw/day (at weeks 26-78) were
    significantly lower than the untreated controls. In contrast,
    animals that had been withdrawn from previous treatment with
    250 mg/kg bw/day for 52 weeks showed improved weight gain during
    the recovery period from week 53 to 78 when compared to animals
    concurrently withdrawn from placebo. Food consumptions were equal
    among treated rats when compared to placebo control. Placebo
    control animals had a significantly lower food consumption compared
    to untreated control rats up to week 78. Eye examinations showed no
    abnormalities related to treatment after 51 and 104 weeks.

    Haematological parameters showed no intergroup differences
    attributable to treatment, whereas clinical chemistry parameters
    revealed an increased plasma cholesterol level, compared with the
    placebo control, in animals treated with 75 and 250 mg/kg bw/day at
    all examinations, and in animals treated with 25 mg/kg bw/day after
    78 and 104 weeks. These alterations were reversible during the
    recovery periods from week 53 to 78 or 79 to 104 in animals that
    had been withdrawn from previous treatment with 75 and 250 mg/kg
    bw/day. No intergroup differences were observed in urine parameters
    with the exception of a light to dark orange/brown discolouration
    of samples collected from a few animals, predominantly at dose
    levels of 75 and 250 mg/kg bw/day at week 51.

         Post-mortem examination revealed a significant increase of
    relative liver weight in animals receiving doses of 75 and
    250 mg/kg bw/day (at week 52 and week 104) and at doses of 5 and
    25 mg/kg bw/day (at week 78) when compared to placebo control.
    However, no intergroup differences attributable to previous
    treatment with 75 or 250 mg/kg bw/day for 52 or 78 weeks were
    measured after recovery periods at sacrifice on week 78 or 104.
    Gross pathology showed an orange discolouration of the skin,
    subcutis and adipose tissues in a number of rats at all dose levels
    and in rats previously treated with 75 and 250 mg/kg bw/day after
    recovery. Discolouration of the liver was seen in a number of
    animals after treatment at a dose level of 25 mg/kg bw/day and
    above, and in a few animals treated with 5 mg/kg bw/day.
    Histopathological examination of the liver showed dose-related
    increased incidence and/or grade of lesions predominantly in
    animals treated at dose levels of 75 and 250 mg/kg bw/day.
    Hepatocyte enlargement was observed in animals receiving
    canthaxanthin at dose levels of 75 and 250 mg/kg bw/day after 52
    and 104 weeks, and in animals given 25 mg/kg bw/day after 52 weeks
    when compared to untreated control or placebo control. A higher
    grade of periportal hepatocyte vacuolation was seen in animals
    treated with 250 mg/kg bw/day from week 52 onwards, whereas a
    higher grade of generalized hepatocyte vacuolation was observed at
    dose levels of 25, 75 and 250 mg/kg bw/day from week 78 onwards
    with signs of a higher grade also among sporadic decedents treated
    with 5 mg/kg bw/day when compared to both control groups. Ground
    glass cells were seen at dose levels of 75 and 250 mg/kg bw/day. A
    higher degree of fat accumulation in hepatocytes was observed at
    250 mg/kg bw/day after 52 weeks, and at 75 and 250 mg/kg bw/day
    from week 78 onward with signs of increased fat accumulation among
    sporadic decedents treated with 5 and 25 mg/kg bw/day. Birefringent
    orange/brown pigment in hepatocytes was observed among animals
    treated at dose levels of 25 mg/kg bw/day and above from week 52
    onwards. At termination of the 26-week recovery period, no
    difference in hepatocyte vacuolation was seen between previously
    treated (75 and 250 mg/kg bw/day) and untreated controls at week 78

    or 104. Ground glass cells were limited to only a few animals
    withdrawn from treatment with 250 mg/kg bw/day after 78 weeks. No
    difference in hepatocyte fat accumulation was apparent between
    previously treated and untreated animals after both recovery
    periods. The hepatocyte pigment in a number of animals was reduced
    when compared to the main group of animals that bad been treated
    with 75 and 250 mg/kg bw/day continuously for 104 weeks. A low
    grade of hepatocyte enlargement was seen in a few animals
    previously treated with 250 mg/kg bw/day for 52 weeks, or 75 and
    250 mg/kg bw/day for 78 weeks. However, hepatocyte enlargement
    was also seen among animals of the untreated control and
    placebo control groups remaining on test up to week 104. A few
    hepatocellular tumours occurred among treated animals. The number
    of benign liver cell tumours were: 1 (5 mg/kg bw/day); 3 (25 mg/kg
    bw/day); and 3 (75 mg/kg bw/day). The numbers of malignant liver
    cell tumours were: 1 (placebo control); 1 (5 mg/kg bw/day); and 1
    (75 mg/kg bw/day). The NOEL in this study was 5 mg/kg bw/day based
    upon the reversibility of liver changes induced at high-dose levels
    (75 and 250 mg/kg bw) and the inconsistency of limited and minimal
    liver findings at the low dose (25 mg/kg bw/day) (Buser, 1992b).

         In a further review of the preceding two long-term studies in
    male rats (Buser, 1992a) and female rats (Buser, 1992b), it was
    stated that clinical as well as most morphological changes observed
    after 1.5 and 1.75 years of treatment with high canthaxanthin doses
    were reversible after a subsequent 0.25 year period, although
    limited elimination of pigment inclusions was observed. In the
    absence of irreversible degenerative processes, it was concluded,
    that the liver effect in male and female rats represented an
    adaptive process (Buser, 1994).

    2.2.1.3  Monkeys

         Groups of 4-11 Cynomolgus monkeys  (Macaca fascicularis) per
    sex (in total 50 males and 49 females, 1-3 years of age) received
    by gavage a water soluble formulation of canthaxanthin at doses of
    0, 0 (placebo), 0.2, 0.6, 1.8, 5.4, 16 or 49 mg/kg bw/day for up to
    3 years. The animals were offered 50-70 g standard primate diet in
    pellets twice daily, fresh fruit twice weekly and one slice of
    bread once weekly. Regular analyses of the diet showed absence or
    insignificant content of aflatoxin B1 and chlorinated
    hydrocarbons.

         As no ophthalmoscopically visible crystalline deposits in the
    retina were observed after one year, 2-4 monkeys/sex/group were
    re-assigned for treatment with canthaxanthin in vegetable oil at
    dose levels of 0 (oil), 200, 500 or 1000 mg/kg bw/day. After 2
    years, 1 male and 1 female treated with 49 and 1000 mg/kg bw/day
    were selected for laser treatment in one eye.

         Interim sacrifice was performed on 1 animal/sex from the
    placebo control group after 1 or 1.5 years, as well as 1 animal/sex
    from the 49 mg/kg bw/day group, after 0.75, 1.0 or 1.5 years. All
    main group animals treated with 0 (7 males and 6 females) or with
    doses from 0.2-49 mg/kg bw/day (4 animals/sex/group, except for one
    pre-terminal decedent in the 0.2, 0.6 and 1.8 mg/kg bw/day groups),
    were sacrificed after 2.5 or 3 years of treatment. At the time of
    submitting the report to WHO, the study was continuing for animals
    receiving doses of 200-1000 mg/kg bw/day and/or on laser treatment
    except for one female receiving 1000 mg/kg bw/day which was
    sacrificed at 2.5 years.

         Observations and examinations performed in all animals during
    the treatment period included morbidity/mortality, clinical signs,
    food consumption, body weights, haematology, clinical chemistry,
    urinalysis, blood levels of canthaxanthin, ophthalmoscopy,
    electroretinography, electrocardiography and cardiovascular blood
    pressure. Post-mortem investigations included organ weights,
    macroscopic pathology and histopathology. The right eye from each
    animal was used for microscopical examination and the left eye for
    chemical analysis.

         One animal each from the 0.2, 0.6 and 1.8 mg/kg bw/day dose
    groups was sacrificed for humane reasons in weeks 145, 147 and 94,
    respectively, whereas 2 animals from the 200 mg/kg bw/day group
    were found dead in weeks 75 and 87 due to pneumonia. The deaths
    were considered to be unrelated to treatment. No signs of
    clinically adverse effects were seen at any dose level. However,
    red-coloured faeces were observed from the first or second day of
    treatment at doses of 5.4 mg/kg bw/day or higher, and a slight to
    marked red-coloured skin was noted at the same dose levels from the
    first or second week of treatment. At 1.8 mg/kg bw/day or lower
    doses, slightly reddened skin was noted in a few animals after one
    year of treatment. No treatment-related effect was seen on food
    consumption, body-weight gain, haematological and clinical chemical
    parameters or on cardiovascular function throughout the treatment
    period.

         Plasma levels of canthaxanthin (all in the  transform)
    monitored at 3-month intervals, were dose-related in groups treated
    with 0.2-49 mg/kg bw/day. Peak levels in each group were seen after
    3 months of treatment, whereas from 1 year onwards, levels were
    consistently lower up to termination of the study. Plasma levels of
    animals receiving 200-1000 mg/kg bw/day from the second year
    onwards were mostly higher but were inconsistent and not
    dose-related.

         Conventional ophthalmoscopy carried out at 3-month intervals
    did not reveal signs of crystalline deposits in the retina of
    animals treated within a dose-range of 0.2-49 mg/kg bw/day or
    200-1000 mg/kg bw/day. However, after almost 3 years, using
    slit-lamp biomicroscopy and wide field lens, isolated single or
    multiple light reflecting spots in the peripheral and central
    retina were observed in 8/18 animals at 200 mg/kg bw/day and higher
    doses, and in laser-treated animals receiving 1000 mg/kg bw/day.
    One animal out of two treated with laser in one eye and given
    49 mg/kg bw/day also showed the presence of light reflecting spots
    in the retina. However, retinographic tests after 1, 2 and 3 years
    provided no evidence of impairment of the visual function at any
    dose level.

         Macroscopic pathology of all animals necropsied during the
    treatment period or after 3 years revealed no lesions or
    abnormalities in any of the organs or tissues that could be
    attributed to treatment. An exception was the orange-red
    discoloration of the GI mucosa and the adipose and connective
    tissue in all canthaxanthin-treated animals. Organ weights of
    animals from treated groups were comparable with those of placebo
    controls. Histopathological changes in the major tissues and organs
    were consistent with findings in historical controls of Cynomolgus
    monkeys. There were no findings of an unusual nature or incidence
    suggestive of systemic target organ toxicity in spontaneous deaths
    or interim and terminally sacrificed animals. Major findings
    included leucocyte and lymphocyte foci, lymphocyte aggregates or
    minor inflammatory lesions in the liver, pancreas, kidney, salivary
    gland, heart, lung and brain or granuloma in the intestinal tract.
    Frozen sections of the liver revealed focal inclusions of
    dark-orange birefringent pigment in a few animals from the 1.8 and
    5.4 mg/kg bw/day groups and in all animals from the 16 and 49 mg/kg
    bw/day groups; no correlation was seen with the lipid content in
    individual livers.

         Microscopic examination of whole-mounts and frozen sections
    of the retina revealed polymorphous birefringent inclusions,
    presumably crystals, in a circular zone of the peripheral retina of
    animals treated with 0.6 mg/kg bw/day or higher, and in the central
    retina of animals treated with 49 mg/kg bw/day or higher after 2.5
    or 3 years. No birefringent inclusions were observed at 0 (placebo
    control) or 0.2 mg/kg bw/day. Birefringent inclusions were also
    demonstrated in animals treated with 49 mg/kg bw/day at the 1 year
    interim sacrifice.

         In polarized light the inclusions were strongly
    light-reflecting and reddish, red/orange to white. In a bright
    field, they were dichroic red/orange to yellow. The size of the
    inclusions was from <1 to 6m. A higher proportion of large
    inclusions was seen with increasing dose. The density of inclusions
    diminished within a zone from 1 to 8 mm distal from the ora
    serrata.

         High density in the periphery and extension of inclusions
    further distal were only seen among animals treated with a dose of
    16 mg/kg bw/day or above. Inclusions were predominantly seen in the
    inner retinal layers e.g., nerve fibre and ganglion cell layer,
    inner plexiform and nuclear layer, and less numerous in the outer
    plexiform layer. In the inner plexiform layer, birefringent
    inclusions were associated with isolated ganglion cells, possibly
    also with amacrine cells, and located inside the perikaryon or
    inside cellular processes. It was not possible to determine the
    precise location of all other birefringent inclusions with the
    techniques used. No inclusions were observed in the outer nuclear
    layer, the rod/cone segment or the pigmented epithelium.

         Total concentration of canthaxanthin (<90%  trans- and
    > 10%  cis-) in the retina revealed considerable variation within
    the individual groups. However, at doses of 0.2 - 49 mg/kg bw/day,
    concentrations of  trans-canthaxanthin were dose-related
    (p = 0.02). The relationship was non-linear indicating saturation
    at the high doses. The mean canthaxanthin concentrations in the
    retina (ng/retina) were 1.4 (placebo control), 6.7 (0.2 mg/kg
    bw/day) and 650 (1000 mg/kg bw/day). Individual canthaxanthin
    concentrations in the retina correlated with individual
    concentrations in plasma over 1.5 and 2 years pre-terminally. The
    mean canthaxanthin plasma concentrations (g/litre) were: 4
    (placebo control), 153 (0.2 mg/kg bw/day) and 7800 (1000 mg/kg
    bw/day). Correlation of canthaxanthin concentrations in the retina
    was also seen with semi-quantitative estimates (polarization
    microscopy) of birefringent inclusions in whole flat-mount or
    cryostat sections of the retina of the contralateral eye.

         The report did not present figures for the concentration of
    the canthaxanthin metabolites 4'-OH-echinenone and isozeaxanthin in
    the retina, but claimed that they were apparently dose-dependent,
    and that the percentages of the metabolites in relation to
    canthaxanthin concentrations in the retina were almost constant.
    The combined amount of lutein and zeaxanthin in the retina was not
    dependent on the concentration of canthaxanthin, which led the
    authors to suggest that canthaxanthin did not affect the
    concentration of the macular carotenoids, lutein and zeaxanthin.

         The authors concluded that prolonged treatment with
    canthaxanthin for up to 2 or 3 years was well tolerated by
    Cynomolgus monkeys, even at very high doses which exceeded intakes
    from food in humans. On the basis of results obtained from the
    study, canthaxanthin did not induce any clinically toxic effect at
    dose levels from 0.2-1000 mg/kg bw/day. Also, no toxic effects were
    seen post-mortem in animals treated with 0.2-49 mg/kg bw/day, or in
    the few animals examined after treatment with 200 and 1000 mg/kg
    bw/day. Clinical and post-mortem observations represented expected
    effects with a carotenoid, such as discolouration of faeces, or the
    dose-related coloration of the digestive tract and organs and
    tissues containing lipids.

         Microscopic crystalline inclusions in the liver and the retina
    in high-dose animals were shown by chemical analysis to be
    associated with the test compound canthaxanthin. However, there was
    no indication of an adverse effect of these deposits on the
    physiological function or morphology of the liver or the eye. The
    NOEL in this study was 0.2 mg/kg bw/day (Buser  et al., 1993,
    1994).

    2.2.2  Special studies on ocular toxicity

    2.2.2.1  In vitro studies

         The formation of canthaxanthin crystals in embryonic chick
    neuronal retina reaggregate cell cultures was studied. In addition,
    the effect of canthaxanthin on lysosomal and mitochondrial
    activity, protein synthesis and differentiation in flat sedimented
    cells of chick embryonic neuronal retina, retinal pigment
    epithelium, brain and meninges were examined. Canthaxanthin was
    added to the cell cultures in association with high density
    lipoprotein which was obtained from chickens fed canthaxanthin or
    placebo. In neuronal retina reaggregate cell cultures, incubation
    with high doses of canthaxanthin resulted in the formation of
    red/brown birefringent entities. The frequency of the birefringent
    entities induced in the cell cultures was directly proportional to
    canthaxanthin concentrations in the medium and occurred at a
    concentration of 1.2 mg/litre of medium and above. Incubation with
    canthaxanthin did not affect the cellular viability and
    differentiation in the cultures (Bruinink  et al. 1992).

    2.2.2.2  Chickens

         Broiler chicks were fed 14.2 g canthaxanthin/kg of diet for 12
    weeks, equal to 28 g/kg bw/day. Histological examination of the
    eyes revealed the presence of birefringent, reddish-brown
    crystal-like structures in the peripheral part of the retina and in
    the uvea of treated animals. Scanning microscopic photometric
    spectrum of crystal-like structures in the retina was similar to

    the spectrum of canthaxanthin reference crystals. No identification
    by chemical analysis of the retinal birefringent material was
    performed (Goralczyk & Weiser, 1992). Scanning microscopic
    photometry would not allow a distinction between canthaxanthin and
    astraxanthin, the latter being a related carotenoid with a similar
    absorption spectrum as for canthaxanthin. Astraxanthin can be
    synthesized by the chicken (Schiedt  et al., 1991).

         A dose-response relationship between ingestion of
    canthaxanthin and the formation of birefringent, crystal-like
    structures in the chick retina was studied. Groups of 4 female
    broiler chicks were fed diets containing 0.2, 0.5, 1.3, 8, 20, or
    50 mg canthaxanthin/kg of feed for 42 days. Dose-dependent
    increased canthaxanthin concentrations were found in retinas,
    plasma and livers by an HPLC technique. In the group fed 8 mg/kg
    feed, equal to 0.5 mg/kg bw/day, microscopic examination of flat
    mount preparations of the retinas under polarized light revealed
    some typical canthaxanthin-related particles. In this group, the
    number of particles correlated highly with canthaxanthin
    concentrations in plasma and less to the concentration in retina.
    The occurrence of particles increased markedly in the groups
    receiving 20 and 50 mg canthaxanthin/kg feed. No retinal particles
    were detected in controls or in the groups fed 0.2, 0.5, or 1.3 mg
    canthaxanthin/kg feed (Goralczyk  et al., 1993).

    2.2.2.3  Guinea-pigs

         Guinea-pigs treated with canthaxanthin at a close level of
    370 mg/kg bw/day for 10 months accumulated canthaxanthin in the
    retina at a concentration of 32 ng/g (Schiedt  et al., 1992).

    2.2.2.4  Ferrets

         Ferrets given 50 mg canthaxanthin/kg bw/day for 12 months did
    not accumulate canthaxanthin in the retina (Schiedt  et al.,
    1992).

         Eighteen ferrets were administered canthaxanthin by gavage in
    an aqueous mixture of water soluble beadlets at a level of 50 mg/kg
    bw/day, 5 days/week, for 12 months. Control animals were fed plain
    beadlets mixed with water. After 12 months of canthaxanthin dosing,
    electroretinograms (ERGs) were measured. Although large variations
    within the groups were observed, the results did not indicate any
    difference between the treated and control groups (Barker & Fox,
    1992).

         In another study in ferrets using a similar dosage regimen as
    by Barker & Fox (1992), canthaxanthin was not detected by an HPLC
    technique in the ferret retinas although the serum level of
    canthaxanthin was 70.2 g/ml at the end of the 12-month period. In
    addition, the concentration of canthaxanthin was 12 and 20 fold
    higher in fat and liver tissues, respectively, than in serum
    (Fox  et al., 1992).

         Microscopical examination of the eyes of ferrets treated with
    canthaxanthin at a dose level of 50 mg/kg bw/day for 24 months did
    not reveal any crystalline deposits in the retina or iris, nor
    choroid or pigmented epithelium. It was concluded that the ferret
    was a less suitable animal model for the study of canthaxanthin-
    induced retinal crystal formation (Goralczyk, 1993).

    2.2.2.5  Monkeys

         An animal model was developed to determine the cause-effect
    relationship and the location of retinal deposits in monkeys
    treated with canthaxanthin. Four monkeys  (Macaca fascicularis)
    were fed canthaxanthin at a daily dose of 11 mg/kg bw/day for 40
    months (total dose 34.5 g). One monkey served as control. Serum
    carotenoids were elevated in all canthaxanthin treated monkeys.
    Predisposing factors to crystal deposition such as glaucoma, venous
    thrombosis and panphotocoagulation were induced in one eye of three
    different experimental monkeys. Ophthalmoscopy, fundus photography
    and fluorescein angiography failed to reveal the classical picture
    of canthaxanthin retinopathy, although a few retinal crystals were
    observed only in the eye with experimentally induced glaucoma.
    However, histological examination revealed birefringent particles
    throughout the retina, from the posterior pole to the periphery, in
    all treated monkeys. The retinal deposits were located in all
    retinal layers, except in the photoreceptor outer segment. It
    was not clear whether the retinal deposits were localized
    intracellularly. No specific cytotoxic effect was found. Contrary
    to humans, in whom retinal crystals accumulate into piles varying
    from 4 to 25 m in diameter, the retinal crystals observed
    histologically in the monkeys were not aggregated and were between
    0.1 to 1 m. It was suggested, that the difference in retinal
    distribution of crystals in monkeys and humans, may account for the
    failure to observe retinal deposits in monkeys by in-vivo
    ophthalmoscopy (Harnois  et al., 1990).

         Schiedt  et al. (1992) compared the concentration of
    canthaxanthin in the retina of monkeys with a reference person who
    had taken sun-tanning pills (16 g in total), was showing retinal
    crystalline deposits and had a concentration of canthaxanthin in
    the retina of 20-30 g/g. The accumulated mean concentration of
    canthaxanthin in the neural retina of 7 monkeys given 49 mg/kg

    bw/day for 36 to 83 weeks (total intake up to 54 g canthaxanthin/
    monkey), was 154 ng/g. The authors calculated that the
    canthaxanthin concentration in retina of the reference person was
    over 100 times higher than that found in the monkey retina, which
    led the authors to assume a higher susceptibility of humans to
    canthaxanthin deposition in the retina.

    2.2.3  Special studies on immune responses

         Canthaxanthin did not show sensitizing effects in the
    guinea-pig optimization test (Geleick & Klecak, 1983).

    2.3  Observations in humans

         The dose-response relationship between retinal crystalline
    deposition and use of canthaxanthin was investigated in a
    retrospective biostatistical study in humans who had taken
    canthaxanthin for either medical or cosmetic reasons. Compiled data
    from published and unpublished reports were analyzed and comprised
    a total of 411 cases of which 95 showed retinal crystalline
    deposition. The daily intake ranged from 15 to 240 mg and the total
    doses varied from 0.6 to 201 g over a period of 1 to 14 years. A
    strong dose-response relationship was demonstrated (p < 0.0001),
    suggesting a NOEL for canthaxanthin crystalline deposits in the
    human retina below a per capita daily intake of 30 mg or a total
    intake of less than 3000 mg (Kpcke  et al., 1994).

         Twenty-seven human subjects (suffering from porphyria) were
    treated with canthaxanthin at dose levels of 15 mg/day for 5 weeks,
    increasing to 60 mg/day for 5 weeks, and subsequently receiving 90
    to 120 mg/day during the summer months. No treatment was given
    during the winter months. Some of the patients received
    canthaxanthin for the first time while others had been treated for
    up to 10 years (total dose up to 170 g). One month dosage of
    15 mg/day canthaxanthin produced no systemic change in the ERG
    scotopic b-wave amplitude while an additional month on a dosage of
    60 mg/day produced a reduction in ERG scotopic b-wave amplitude
    which was more pronounced after a further month at a dose of
    90 mg/day. Human subjects with canthaxanthin crystals in the retina
    showed an even more marked reduction in the ERG scotopic b-wave
    amplitude. However, the reduction in the ERG scotopic b-wave
    amplitude was not correlated with the concentration of
    canthaxanthin in blood. During winter time (off treatment), the
    effect on the ERG scotopic b-wave amplitude was reversible. It was
    suggested that the mechanism for the reduction of the ERG scotopic
    b-wave amplitude was due to the concentration of canthaxanthin by
    the Mller cells, known to generate the scotopic b-wave. The NOEL
    in this study was 15 mg/day, equivalent to 0.25 mg/kg bw/day
    (Arden  et al., 1989).

         The visual function was assessed by means of threshold static
    perimetry on 19 patients who had ingested canthaxanthin (amount
    ingested not given); 11 had maculopathy and 8 did not. Patients
    with no history of canthaxanthin ingestion served as controls. All
    patients had visual acuity of 6/9 or better. Threshold static
    perimetry was re-evaluated 2 to 3 years after cessation of
    canthaxanthin ingestion. For both testing sessions, patients with
    retinal deposits presented lower retinal sensitivity than controls,
    while patients without retinopathy did not differ significantly
    from the control group. The results led the authors to suggest,
    that canthaxanthin retinopathy adversely affected the neurosensory
    retina (Harnois  et al., 1988).

         Reversibility of canthaxanthin retinal deposits was observed
    in 14 patients treated with cumulative doses of canthaxanthin of up
    to 178 g for up to 12 years. Up to 70% reduction in the number of
    retinal deposits was observed 5 years after discontinuation of
    treatment (Leyon  et al., 1990).

         Canthaxanthin-related carotenoids, present in the human and
    primate retinal macula region, were identified to be lutein and
    zeaxanthin (Handelman  et al., 1991; Handelman  et al., 1988). In
    humans, the dominant carotinoid in the macula region was
    zeaxanthin, whereas lutein was dispersed throughout the entire
    retina (Handelman  et al., 1988).

         No signs of hepatotoxicity (tests not described) were evident
    in 11 patients, 10 to 61 years old who had been treated against
    erythropoietic protoporphyria with canthaxanthin at cumulative
    doses ranging from 3 to 150 g over a period of 1 to 12 years
    (Norris & Hawk, 1990).

    3.  COMMENTS

         Since the last review, several studies have been conducted in
    order to identify a suitable animal model for the deposition of
    canthaxanthin crystals in the retina. In Cynomolgus monkeys,
    feeding with canthaxanthin for 2.5 years resulted in a
    dose-dependent accumulation of this substance in the retina.
    Although not visible by conventional ophthalmoscopy, birefringent
    inclusions were observed microscopically in the inner retinal
    layers, with a distribution similar to that seen in human
    canthaxanthin retinopathy. The NOEL in this study was 0.2 mg/kg
    bw/day.

         A dose-response relationship between canthaxanthin intake and
    the development of crystalline deposits in the retina of humans had
    not previously been definitely established. However, a
    comprehensive retrospective biostatistical study of both
    unpublished and published studies, which included data on total
    intake ranging from 0.6 to 201 g over a period of 1-14 years,
    showed a strong dose-response relationship, suggesting a NOEL for
    canthaxanthin crystalline deposits in the human retina below a
    daily intake of 30 mg canthaxanthin per person.

         In 27 human subjects, some of whom received canthaxanthin for
    the first time while others had been treated for up to 10 years, no
    impairment of vision, as measured by electroretinography as a
    reduction in the scotopic B-wave amplitude, was observed at a daily
    intake of 15 mg of thaxanthin per person (equivalent to 0.25 mg/kg
    bw/day) over a period of 5 weeks. An additional month on a dosage
    of 60 mg/person/day produced a reduction in scotopic B-wave
    amplitude, which was more pronounced after a further month of
    treatment with 90 mg of canthaxanthin/person/day.

         Additional long-term toxicity/carcinogenicity studies in rats
    confirmed that canthaxanthin, as previously observed, was hepatoxic
    in this species, but provided no evidence of carcinogenicity. At
    low doses (5 or 25 mg/kg bw/day) only sporadic occurrence of
    vacuolated liver cells was observed, and at higher dose levels
    (75 or 250 mg/kg bw/day) this change appeared reversible. The NOELs
    were 5 and 25 mg/kg bw/day in female and male rats, respectively.
    In contrast to the liver cell changes observed in rats, no such
    changes were seen in monkeys given up to 49 mg of canthaxanthin/kg
    bw/day for up to 2.5 years.

         Hepatotoxicity in humans due to ingestion of canthaxanthin has
    not been reported and, although the number of cases was limited, no
    signs of hepatotoxicity were seen in patients with erythropoietic
    protoporphyria treated with a total of 3-150 g canthaxanthin over
    a period of 1-12 years.

    4.  EVALUATION

         The Committee allocated an ADI of 0-0.03 mg/kg bw to
    canthaxanthin, based on a NOEL of 0.25 mg/kg bw/day in humans and
    a safety factor of 10.

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    See Also:
       Toxicological Abbreviations
       Canthaxanthin (WHO Food Additives Series 22)
       Canthaxanthin (WHO Food Additives Series 26)
       Canthaxanthin (WHO Food Additives Series 44)
       CANTHAXANTHIN (JECFA Evaluation)