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 µg
equivalent 14C-canthaxanthin/g at the high dose), with moderate
levels in the spleen, liver, bone marrow, skin, and fat (0.1-0.9 µg
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 µg 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 µg/g and 0.13 µg/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 6µm. 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 (µg/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 µg/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 µm in diameter, the retinal crystals observed
histologically in the monkeys were not aggregated and were between
0.1 to 1 µm. 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 µg/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 (Köpcke 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 Müller 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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