CANTHAXANTHIN
1. EXPLANATION
Canthaxanthin is a diketo carotenoid pigment with an orange-red
colour. It occurs in the edible fungus, chanterelle (Cantharellus
cinnabarinus), in the plumage and organs of flamingoes, the scarlet
ibis (Guara rubra) and the roseate spoonbill (Ajaja ajaja), and in
various crustacea and fish (trout, salmon) (Haxo, 1950; Fox 1962a, b;
Thommen & Wackernagel, 1963).
Canthaxanthin has previously been evaluated for an acceptable
daily intake at the tenth, eighteenth and thirty-first meetings of the
Committee (Annex 1, references 13, 35, 77). 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 the the following:
1. further details of the long-term studies in rats and mice, for
which summary data were submitted, including ophthalmological
data where available;
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.
Since the previous evaluation, further data have become available
and are summarized and discussed in the following monograph. The
previously published monographs have been expanded and are
incorporated into this monograph.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1. Absorption, distribution, metabolism and excretion
2.1.1.1 Rats
Adult rats were fed a range of oral doses of canthaxanthin (doses
not specified) for 13 and 20 weeks respectively. Canthaxanthin
accumulated in fat and some organs, notably the liver and spleen.
Only slight depletion of canthaxanthin from fat occurred over a period
of 1 month, indicating very slow elimination from this tissue
(Hoffmann-La Roche, 1986).
Groups of rats were given daily doses of 0.6, 6 or 60 mg
canthaxanthin/kg bw daily for five weeks. Highest organ
concentrations were found in the liver and spleen, the tissue levels
corresponding to the three dietary dose levels being 0.9, 12 and 125
µg/g in liver and 2.6, 50 and 67 µg/g in spleen, respectively. Levels
in other organs were much lower (0.2-1.5 µg/g at the highest dose
level). After discontinuing administration of canthaxanthin, tissue
levels in the adrenals and small intestine fell to one-quarter and to
one-third of their original levels over a period of 2 weeks (adrenals)
or 1 month (intestine) (Hoffmann-La Roche, 1986).
When rats were fed daily doses of 50-60 mg canthaxanthin/kg bw
for 9 weeks, the concentration in the eye remained at approximately
0.1 µg/g with no further accumulation. After administration of doses
1.2, 2.0, 3.4, 5.6, 9.8, 16.7 or 28.4 ppm canthaxanthin (equivalent to
1.4 mg/kg bw/day at the top-dose level) for 20 weeks, maximum
concentrations in the eye were found to be about 0.01 µg/g; the
residual levels in the eye fell to 0.002 µg/g over a 4-week period
after removal of canthaxanthin from the diet (Hoffmann-La Roche,
1986).
Groups of 40 male rats were fed diets containing canthaxanthin at
concentrations of 1.2, 3.4, 5.8, 9.8, 16.7 or 28.4 ppm. After 96 days
8 animals from each dose group were killed and the following tissues
analyzed for canthaxanthin: plasma, liver, spleen, kidney, heart,
testicular fat, adrenal fat, intestine, cerebellum, cerebrum, adrenal
gland, thyroid and eyes. A further 8 animals from each dose group
were maintained on canthaxanthin-free diet for periods of 7, 15 and 29
days respectively prior to analysis. The remaining 8 animals per dose
group were maintained on the test diets for a total of 137 days before
examination.
Tissue levels of canthaxanthin were more dependent on dietary
concentration than duration of exposure over the period of 98 to 136
days. The highest concentrations reported in tissues in the highest
dose group were found in spleen (3.2 µg/g), small intestine
(2.63 µg/g), liver (1.56 µg/g) and adipose tissue (0.79-0.91 µg/g);
levels in cerebrum, thyroid and eyes were low and close to the
detection limit of the assay. Depletion of canthaxanthin was obvious
in most tissues during the withdrawal period except for fat, where
levels fell slowly if at all. In the spleen there was a rapid fall
during the first seven days after withdrawal to about 20% of the
original concentration but thereafter the depletion was very slow.
Conversely, liver concentrations fell quite steeply and continuously
over the depletion period. Because the maximum levels achieved were
so low in brain, thyroid and eyes, it was not possible to follow the
course of depletion in these tissues with any accuracy (Bausch
et al., 1987); this appears to be a more complete report of the
preceding summary (Hoffmann-La Roche, 1986).
The distribution of the radioactivity derived from 6,7,6',7'-
14C-canthaxanthin was studied in male rats after oral administration
of 318-372 nmoles in about 2 ml of a liposomal preparation. At
intervals of 4, 24, 48, 96 and 168 hours after dosing, two animals
were sacrificed and radioactivity was measured in liver, spleen,
heart, lungs, thymus, kidneys, adrenals, testes, epididymes, skin,
eyes and brain. Samples were also taken from muscle, fat, pancreas,
plasma and erythrocytes, and the contents of the stomach, small
intestine and colon (including cecum) were also counted separately.
On the assumption that enterohepatic circulation had not occurred, the
amount of the dose absorbed was calculated to be about 8% (range
4-11%), based on the amount in the tissues and the amount excreted in
urine.
The distribution of the absorbed radioactivity was dependent on
time after dosing. Excluding the gastrointestinal tract and its
contents, the highest levels of radioactivity were found in the liver.
Levels in the tissues were not reported if these were less than 1% of
the dose, except for the eyes where the content never exceeded 0.05%
of the dose. After 24 hours, about 16% of the administered dose
remained in the body (including gastrointestinal contents) and this
fell to 0.3% after 7 days. It was calculated that, of the absorbed
radioactivity (assuming no enterohepatic circulation), about half was
excreted in the urine in 24h and 96% in 7 days (Glatzle & Bausch,
1988a).
When rats were fed a diet containing 10 ppm canthaxanthin for
13-14 weeks, the mean renal fat level was 0.43 µg/g, falling to
0.29 µg/g during the four weeks after the cessation of exposure. When
administered at a dietary concentration of 100 ppm for 31 weeks,
canthaxanthin levels in renal fat were 4.3 µg/g and fell to 2.2 µg/g
over a withdrawal period of 31 weeks. This study illustrates the very
long half-life of canthaxanthin in adipose tissue (Glatzle & Bausch,
1988b).
2.1.1.2 Chickens
Canthaxanthin was reported earlier not to exhibit provitamin A
activity (Hoffmann-La Roche, 1966). However, in recent studies in
chickens, canthaxanthin was shown to be converted to vitamin A in
small amounts. Groups of 15 broiler chickens, 37 days old, were given
diets containing 8.9, 18 or 35 ppm cantaxanthin along with 0, 300 or
600 I.U. vitamin A/kg feed. At each dietary level of vitamin A,
canthaxanthin caused a dose-related increase in plasma and liver
concentrations of both vitamin A and canthaxanthin. The lowest level
of canthaxanthin, in the absence of dietary vitamin A, yielded higher
plasma and liver vitamin A levels than did a diet containing 600 I.U.
vitamin A/kg in the absence of canthaxanthin (Hoffmann-La Roche,
1986).
When fed to chickens at a concentration of 70 ppm in a diet
otherwise free of carotenoids, canthaxanthin was partially reduced to
4-hydroxy-echinenone, with further reduction to isozeaxanthin. Both
metabolites and their ester derivatives were detected, together with
unchanged canthaxanthin, in various tissues including intestinal
mucosa, liver, serum, skin, claws, and feathers (Tyczkowski et al.,
1988).
Two groups of 5 week old chickens were given diets containing 600
I.U. or 12,000 I.U. vitamin A/kg; both groups were also given the
equivalent of 10 mg 14C-labelled canthaxanthin/kg diet, in gelatin
capsules, daily for four weeks. At the end of this period,
canthaxanthin administration ceased and one bird from each group was
sacrificed after 0, l, 6, 10, 13 and 21 days.
The total retention of canthaxanthin in the body at the end of
the dosing period was approximately 6% in the low vitamin A group and
10% in the high vitamin A group. The radioactivity was similarly
distributed in both groups, most of the retained activity being found
in blood (approx. 20%), muscle (20-24%) feathers (approx. 13%) and
skin (8-12%). The highest tissue concentrations (as canthaxanthin
equivalents according to radioactivity) were found in the retina
(14-27 µg/g), uropygial gland (13-35 µg/g/), bile (approx. 10 µg/g),
toe-web (10-20 µg/g) and, in birds of the high vitamin A group, liver
(11-13 µg/kg). There was a continuous fall in the concentration of
radioactivity in most tissues except for fat, feathers and retina
after the cessation of exposure. In fat, activity fell during the
first 10 days of depletion and then plateaued. With the small number
of birds it was not possible to demonstrate any decrease in activity
in the retina, the retinal activity of the high vitamin A group being
similar after 0 and 15 days.
The metabolites 4'-hydroxy-echinenone (4HE) and isozeaxanthin
(IZX) were identified in muscle, liver and duodenum. Conversion of
canthazanthin to retinol occurred in the liver and duodenum, the main
intermediates detected being 4-oxo-retinal and 4-Oxo-retinol.
4-xo-12'-apo-ß-carotenal was also identified as a metabolite (Schiedt
et al., 1988). Canthaxanthin can thus act as a precursor of vitamin
A in the chicken (Schiedt, 1987).
The distribution of canthaxanthin fed to laying hens differs from
that of broilers; 30-40% of the dose was deposited in the egg yolk, 7%
in body tissues and 42-54% was excreted. During a 10-day depletion
period after withdrawal of canthaxanthin, the body stores were
reported to be mobilized and excreted via eggs and excrement;
concentrations of canthaxanthin in fat were stated to be low and to
remain constant (Hoffman-La Roche, 1986).
15,15'-3H-Canthaxanthin was fed to two groups of 4 laying hens
at dietary levels of 4 and 8 ppm, respectively, for up to 4 weeks
followed by a 10 day withdrawal period. One bird from each group was
sacrificed after 15 days, two birds from each group after 29 days and
one bird from each group after the withdrawal period. During the
period 15-28 days, about 40% of the ingested activity appeared in the
egg yolk at concentrations (canthaxanthin equivalents) of 14 and
29.3 µg/g respectively in the low and high dose groups. Of the
3H-labelled carotenoids in the yolk, about 97% was unchanged
canthaxanthin, 2.3% was 4HE and 0.4% was IZX. Only 0.2-1% of residual
radioactivity was found in the pooled spleens and kidneys of all eight
birds, of which canthaxanthin accounted for 60% and 80% in the
respective organs. 4HE and IZX accounted for 6% and 5%, respectively,
of the activity in the spleen and the remaining 28% was unidentified
(because of the low residual amounts present); the corresponding
figures for the kidneys were 6%, 2% and 10%, respectively. After 28
days, canthaxanthin was present in peritoneal fat mainly unchanged at
a concentration of 0.5 µg/g. In the liver, about 60% of the
radioactivity was present as metabolites, mainly 4-oxo- and 4-hydroxy-
retinol, and retinol, confirming that canthaxanthin can act as a
precursor to vitamin A in the hen (Schiedt & Mayer, 1986).
Day old chicks received a vitamin A-free diet supplemented with
9, 18 or 36 ppm canthaxanthin and with or without small vitamin A
doses of 300 or 600 IU/kg for 40 days. In each case, canthaxanthin
caused a dose-dependent growth promotion, better feed conversion and
increased vitamin A levels in plasma and liver. In chicks receiving
600 I.U. vitamin A together with 0, 9 or 18 ppm canthaxanthin in the
diet, the respective duodenal mucosal activities of ß-carotene-15,15'-
dioxygenase were 74, 142 and 119 pmoles retinal/h/mg protein (Weiser
et al., 1987).
2.1.1.3 Dogs
The tissue distribution of canthaxanthin was investigated in dogs
which had received 50, 100 or 250 mg/kg bw/day canthaxanthin for 52
weeks, corresponding to total doses of 200, 400 or l,100 g of
canthaxanthin, respectively. The highest mean tissue concentration
was seen in adipose tissue (24 µg/g in the top dose group).
Relatively high concentrations were also seen in the adrenals
(15.1 µg/g), skin (9.62 µg/g) and liver (8.1 µg/g/) in the low-dose
group. The total amount of canthaxanthin extracted from 8 eyes of
treated dogs was 0.1-0.4 µg, but ophthalmological examinations were
not reported, so it was not possible to determine whether crystalline
deposits had formed (Hoffmann-La Roche, 1986).
2.1.1.4 Humans
A pharmacokinetic study was performed in which plasma levels of
canthaxanthin were measured at intervals after multiple dosing of
human volunteers. Ten subjects were each given 1 mg canthaxanthin 6
times a day for 5 days, corresponding to a total dose of 30 mg. A
further ten subjects received 8 mg canthaxanthin 6 times a day for 2
days, for a total dose 96 mg. Blood samples were taken at the start
and at 12 hour intervals for 8 days, and canthaxanthin concentrations
were determined by HPLC. The elimination half-life was calculated as
4.5 days in each group and the proportion of the dose absorbed was
estimated to be 12 and 9% in each group, respectively. The calculated
steady state plasma concentrations of canthaxanthin after daily
ingestion of 6 mg (6 times 1 mg) or 48 mg (6 times 8 mg) were 1,843 or
10,346 µg/1, respectively (Kubler, 1986).
In a more detailed report of the above studies it was concluded
that canthaxanthin was cleared from serum with a half-life of 5.3
days, and after administration in multiple oral doses, steady-state
serum concentrations were achieved after approximately 48 hours. The
absorption of canthaxanthin is incomplete, no more than 34% of a 1 mg
oral daily dose being absorbed, with the proportion of the dose
absorbed falling with increasing dose. About 60% of the absorbed dose
is transferred to fat tissue and remobilization is unlikely even under
conditions of rapid fat utilization. An intake of 30 mg
canthaxanthin/day would lead to steady-state blood concentrations of
approximately 6 mg/1 and a daily ingestion of 3.5 mg canthaxanthin,
equivalent to the temporary ADI for a 70 kg person, would lead to a
steady-state concentration of 1.1 mg/1 (Schalch, 1988a).
2.2 Toxicological studies
2.2.1 Acute toxicity
Species Route LD50 Reference
(mg/kg bw)
Mouse oral 10,000 Hoffmann-La Roche,
1966
2.2.2 Short-term studies
2.2.2.1 Mice
Canthaxanthin was fed to groups of 10 male and 10 female albino
outbred mice at doses of 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day
for 13 weeks. Males and females of the two highest dose groups showed
the lowest body weight throughout, but the differences were not always
statistically significant and in some cases they correlated with lower
food intake. Apart from a red discolour ation of some internal
organs, no adverse effects were seen at autopsy and histological
examination of the two highest dose groups and controls did not show
any treatment related effects (Steiger, 1981).
2.2.2.2 Rats
Canthaxanthin was fed to groups of 10 male and 10 female albino
outbred rats at dose levels of 0, 125, 250 500, 1000 and 2000 mg/kg
bw/day for 13 weeks. Organ function tests (BSP retention and phenol
red elimination) were carried out at weeks 12 and 10, respectively, on
animals in the control and top dose groups; comprehensive urinalysis
was performed on all groups during week 11. At termination, detailed
ophthalmological, hematological and clinical chemical examinations
were carried out, and a complete histological examination was
performed on rats in the control and the top two dose groups.
A small decrement of weight gain relative to controls was seen in
animals of both sexes in the highest dose group only. Values for
plasma cholesterol were higher than controls in all treated groups but
were within normal limits. With this exception, all the other
clinical, chemical and hematological parameters were unaffected by
treatment and no abnormalities were detected in the organ function
tests. At autopsy, no gross abnormalities due to treatment were
observed other than a red or orange discolouration of feces and some
internal tissues. Kidney weights of males from the highest dose group
were significantly reduced relative to controls and the weights of
adrenals from females of the 1000 mg/kg bw/day dose group also were
reduced relative to placebo controls. No treatment-related
pathological effects were seen on histological examination (Steiger &
Buser, 1982).
2.2.2.3 Dogs
Canthaxanthin was fed to groups of 3 male and 3 female beagle
dogs at dose levels of 0, 250 or 500 mg/kg bw/day for 13 weeks. There
were no effects on food and water intake or on body weight gain.
Apart from red/orange staining of the feet, muzzle, abdominal fat and
the feces, there were no clinical signs related to treatment and
ophthalmoscopic examination did not reveal any abnormalities related
to the test compound. At termination, organ weights were within
normal limits and there were no histological abnormalities
attributable to treatment (Chesterman et al., 1979).
2.2.3 Long/term carcinogenicity studies
2.2.3.1 Mice
In a preliminary report of an 80-week study in mice in which the
animals received 0, 250, 500 or 1000 mg canthaxanthin/kg bw/day it was
stated that no signs of systemic toxicity and no changes of tumour
incidence were seen that could be related to treatment (Hoffmann-La
Roche, 1966).
When given orally, canthaxanthin exhibited no promotional
activity in mice treated dermally with dimethylbenz(a)anthracene or
benzo(a)pyrene (Mathews-Roth, 1982; Santamaria et al., 1982).
Oral doses of 6680 mg canthaxanthin/kg bw/day gave some
protection against the skin carcinogenicity of regular exposure to UV
radiation (Mathews-Roth, 1982).
Groups of 60 male and 60 female CD-1 mice, approximately 4 weeks
old, were given canthaxanthin by incorporation in the diet at levels
calculated to achieve doses of 0, 0(placebo control), 250, 500 or 1000
mg/kg bw/day for 90 weeks (males) or 98 weeks (females). The
canthaxanthin was microencapsulated in water-soluble beadlets
containing 10% canthaxanthin and similar beadlets devoid of
canthaxanthin ("placebo beadlets") also were prepared. The test
animals received a constant dietary concentration of beadlets, the
different dose levels being achieved by mixing appropriate proportions
of the canthaxanthin- and placebo-beadlets; one control group received
basal diet and the second (placebo) control group received a similar
concentration of placebo beadlets as was given to the test animals.
Throughout the study, the achieved intakes of canthaxanthin
approximated the nominal value. Ten animals of each sex per dose
group were sacrificed after 52 weeks for interim examination.
Food intake and body weight gain were similar in treated and
placebo control mice throughout the study. No clinical abnormalities
other than a reddish discolouration of feces, fur and skin were
observed and ophthalmoscopic examination did not reveal any
abnormalities. There was no indication of any treatment-related
effect on survival and no factors attributable to treatment were among
the assignable causes of premature death. No treatment-related
hematological abnormalities were observed and the only significant
biochemical change was a higher blood cholesterol level in all groups
of treated animals of both sexes at week 52 and females at week 98
compared to placebo controls. At termination, there were no effects
of treatment on organ weights and the only gross observations at
autopsy related to dosing were a generalized orange discolouration of
fur/skin, subcutis, adipose tissue and gastrointestinal tract.
Histopathological examination did not reveal any treatment-related
effects on the incidence of any tumour type or on the total number of
tumours per group. Lipid positive granules were observed in the
sinusoidal cells in the liver of all mice treated with canthaxanthin,
the incidence varying in a dose-related manner. Orange-brown pigment
was present in sinusoidal cells and, to a lesser degree in macrophages
and some hepatocytes, in treated animals. Other histopathological
findings were not treatment-related and were within the normal
background range for mice of this strain and age (Rose et al.,
1987).
2.2.3.2 Rats
Groups of 25-30 male and female rats received 0, 0.5, 2 or 5%
canthaxanthin in their diets for 93-98 weeks. No adverse effects were
noted on food consumption or weight gain. Mortality and tumour
incidence were not increased (Hoffmann-La Roche, 1966).
Groups of 70 male and 70 female weanling CD Sprague-Dawley rats
were given canthaxanthin by incorporation in the diet at levels
calculated to achieve doses of 0, 0(placebo), 250, 500 or 1000 mg/kg
bw/day for up to 104 weeks. The canthaxanthin was microencapsulated
in water-soluble beadlets containing 10% canthaxanthin and similar
beadlets devoid of canthaxanthin ("placebo beadlets") also were
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 basal diet and the second (placebo) control
group received a similar concentration of placebo beadlets as was
given to the test animals. The achieved dose was about 90% of the
target dose for males while for females it was 100-10l% of the target.
Interim sacrifices of 10 males and 10 females of each group were
carried out at 52 weeks and a further 3 or 4 animals of each sex per
group after 78 weeks. A further 10 animals of each sex per dose group
were withdrawn and placed on a normal untreated diet at 78 weeks and
maintained on this diet for up to 16 weeks (males) or 20 weeks
(females).
Overall food and water intake and body weight gain of placebo
control and canthaxanthin-treated animals were significantly lower
than untreated controls; body weight gain of canthaxanthin-treated
animals was lower than that of placebo controls, especially for
females, but did not vary in a dose-related way between dose groups.
Body weight gain of previously treated animals and placebo controls
increased to a similar extent during the withdrawal period and it was
concluded that the reduced food intake and body weight gain resulted
from incorporation of the beadlets into the diet rather than from an
effect of canthaxanthin per se. No clinical abnormalities other than
reddish discolouration of feces, fur and skin were observed and
ophthalmoscopic examination did not reveal any abnormalities. The
mortality among placebo and canthaxanthin-treated animals was much
lower than in untreated controls. The prolongevity was attributed to
the reduced food intake resulting in leaner, healthier animals. No
treatment-related effects were seen in the hematological or urine
analytical examinations. The clinical biochemical examinations
revealed treatment-related increases in the following parameters in
females only: AP and SGPT (weeks 12, 26, 52 and 78), SGOT and
cholesterol (weeks 12, 26, 52, 78 and 104), gamma-GT (weeks 52, 78 and
104) and bilirubin (weeks 26, 52, 78 and 104).
At the interim sacrifices and at termination, liver weights of
female rats receiving canthaxanthin were greater than those of placebo
or untreated controls; no other treatment-related organ weight changes
were observed. Gross pathological examination at termination revealed
orange discolouration of liver, intestinal contents, skin, fur,
subcutis, adipose tissue and extremities in treated animals in all
dose groups. Histological examination of animals sacrificed at the
interim and terminal sacrifices revealed treatment-related lesions
only in the liver. Male and female animals from all treatment groups
showed hepatocyte enlargement and brown pigment deposition. Females
from all dose groups additionally showed eosinophilic hepatocellular
foci, hepatocyte vacuolation, bile duct hyperplasia and cystic bile
ducts; a few females from the top two dose groups also showed
cholangic fibrosis and foamy macrophages. At the terminal kill, there
was a higher incidence of benign liver nodules in females of all
treatment groups but this was not dose-related. It was not possible
to establish a no-observed-effect level in female animals in this
study in relation to hepatic biochemical or morphological changes
(Rose et al., 1988).
In a further review of the preceding study, a re-evaluation of
the histological data and diagnoses and statistical analysis of
non-neoplastic and neoplastic liver lesions in males and females was
performed. It was concluded that a toxic effect to the liver was seen
at all dose levels in this long-term study. Trend tests showed that
the effect was much more pronounced in females, the dose-related
response was flat, and no threshold dose could be determined for
either sex. The incidence of liver nodules was slightly increased in
females but was limited to benign tumours only; there was no
indication of an increase in malignant tumours (Buser & Banken, 1988).
2.2.3.3 Dogs
Oral doses of 0, 50, 100 or 250 mg canthaxanthin/kg bw/day for 52
weeks were well tolerated by dogs and no adverse effects attributable
to treatment were observed (Hoffmann-La Roche, 1986).
Groups of 4 male and 4 female beagle dogs were given
canthaxanthin in beadlets in the diet at doses of 0, 0(placebo), 50,
100 or 250 mg/kg bw/day for 52 weeks. Canthaxanthin was micro-
encapsulated in water-soluble beadlets in the same manner as described
in the summaries of the studies in mice (section 2.2.3.1) and rats
(section 2.2.3.2). Detailed hematological examinations and blood
biochemical and urine analyses were performed at weeks 12, 24, 39 and
51 at which times an ophthalmoscopic examination also was carried out.
There were no deaths and no indicative signs of systemic toxicity
throughout, and there were no adverse effects of treatment on food
intake and body weight. Ophthalmoscopy did not detect any
abnormalities due to the test material. No significant intergroup
differences in the hematological parameters indicative of a treatment-
related effect were observed at any time. Group mean values for the
biochemical parameters showed occasional significant differences from
placebo controls but these were not systemic or dose-related and were
generally within normally acceptable limits. Urinalysis did not
reveal any treatment-related differences in any parameter measured.
At autopsy, reddish discolouration was observed in fur and hair,
adipose tissue, aortic valve and right atrioventricular valve of
selected animals; a cherry red discolouration was seen in the liver of
one animal only from each of the 50 and 100 mg/kg/bw/day dose groups.
Organ weights were not affected by treatment. Detailed
histopathological examination did not reveal any dose-related
pathological effects. Moderate amounts of dark pigment seen in
midzonal hepatocytes and in some Kupffer cells of one female from the
50 mg/kg bw/day group and one male from the 100 mg/kg bw/day group
were attributed to treatment but no such pigmentation was seen in the
highest dose group (Harling et al., 1987).
2.2.4 Reproduction studies
2.2.4.1 Rats
In a three-generation reproduction study, canthaxanthin was given
to male and female rats at doses of 0, 0(placebo), 250, 500 and 1000
mg/kg bw/day in the form of beadlets in the diet throughout three
generations. Canthaxanthin was micro-encapsulated in water-soluble
beadlets in the same manner as described in the summaries of the
studies in mice (section 2.2.3.1) and rats (section 2.2.3.2). In each
generation, two litters were obtained and the second and third
generations were produced from parents derived from the first litter
in the F1 and F2 generations, respectively.
There were no adverse effects of treatment or mating performance,
duration of gestation, parturition or ability of dams to lactate and
rear their offspring successfully. Some treatment-related effects
without consequences for reproductive performance were noted in the
form of:
a) orange/red colouration of feces, fur, viscera and adipose
tissue;
b) reduced food efficiency earlier than in placebo controls and
a tendency for the growth curve to plateau at a lower value;
c) markedly increased levels of SAP, SGPT, SGOT and cholesterol
in adult females;
d) significantly increased liver weights in culled weanlings in
each of the six litters;
e) histological changes in the liver with foci of foamy
macrophages in the liver sinusoids of F0 and F2 adult
females and slightly increased hepatocyte vacuolation in F2
adults at 500 and 1000 mg/kg bw/day;
f) decreased adrenal weight in adult females at all doses;
g) increased spleen weight in adult females of the top dose
group.
The above adverse effects were partially reversed during an
eight-week withdrawal phase at the end of the study (Bottomley
et al., 1987).
2.2.5 Special studies on embryo/fetotoxicity
2.2.5.1 Rats
Groups of 40 pregnant FU-Albino rats were given canthaxanthin in
the diet at dose levels of 0, 250, 500 or 1000 mg/kg bw/day on days 7
to 16 of pregnancy, inclusive. On the 21st day of gestation the dams
of each group were divided into two subgroups, designated a necropsy
subgroup and a rearing subgroup.
Animals in the necropsy subgroups were sacrificed at day 21 and
the uteri examined for the number and location of implantations and
resorptions; the number of corpora lutea were counted and the fetuses
were examined macroscopically, weighed and measured. The fetuses of
10 litters per dose group were examined for skeletal abnormalities;
the fetuses of 7 litters were examined for soft tissue defects.
Animals in the rearing subgroups were allowed to litter
spontaneously and rear their young to weaning. Litter size and
weights, and maternal weights were recorded on days 1, 4, 12 and 23
post partum. At day 23 the offspring of 8 litters per group were
necropsied and heart, liver and kidney weights were measured.
No treatment-related effects were seen on the dams, and there was
no indication of any embryotoxic or teratogenic action of
canthaxanthin at any of the dose levels used. The rearing experiment
showed no evidence of effects on lactation nor of functional
abnormalities in the offspring (Kistler, 1982).
2.2.5.2 Rabbits
Groups of 20 mated female Swiss hare rabbits were given doses of
0, 100, 200 or 400 mg canthaxanthin/kg bw/day by gavage in rape seed
oil on days 7 to 19 of pregnancy inclusive. All the dams were killed
on day 30 of gestation and examined for the number and location of
implantations and resorptions; the number of corpora lutea were
counted. The fetuses were examined macroscopically, weighed and
measured, then tested for 24h viability in an incubator at 34°C. All
the fetuses were then dissected and examined for soft tissue
abnormalities. All the fetuses were X-rayed and those which could not
be judged definitely by radiography were cleared and stained with
Alizarin red for skeletal examination.
The test compound was well tolerated and no effects were seen on
maternal body weight gain in any dose group. The reproductive
parameters measured were within the range of concurrent controls; a
slight but statistically significant increase in resorptions was noted
in the 100 mg/kg bw/day dose group but there was no such effect in the
higher dose groups and it was considered not to be treatment-related.
Sporadic malformations of different types occurred in a few fetuses of
all groups, including controls; because of their single occurrence and
distribution in all groups, these abnormalities were not attributed to
treatment. In all treatment groups skeletal anomalies were in the
control range. It was concluded that, under the conditions of the
study, canthaxanthin was neither embryotoxic nor teratogenic
(Eckhardt, 1982).
2.2.5 Special studies on genotoxicity
Canthaxanthin did not induce mutations in Salmonella typhimurium
strains TA98, TA100, TA1535, TA1537 or TA1538 (Chételat, 1981) nor in
Saccharomyces cerevisiae (Chételat, 1986).
Canthaxanthin was negative in a mouse micronucleus assay after
two-fold application of up to 222 mg/kg bw (Gallandre 1980),
Canthaxanthin, in the presence of an activating system did not
induce mutations to 6-thioguanine-resistance in V79 Chinese hamster
lung cells (Strobel, 1986) and did not induce DNA damage resulting in
unscheduled DNA synthesis in primary cultures of rat hepatocytes
(Strobel, 1986).
2.2.6 Special studies on effects on the immune response
Male Wistar rats were fed diets containing either 2 g/kg
ß-carotene or canthaxanthin, or basal diet for up to 66 weeks.
in vitro immune responses of splenocytes to T- and B-lymphocyte
mitogens were determined. T- and B-lymphocyte responses were enhanced
in the groups fed ß-carotene or canthaxanthin (Bendich & Shapiro,
1986).
2.2.7 Special studies on ocular toxicity
(see also Human Studies)
2.2.7.1 Rabbits
Preliminary results of studies in rabbits on the ocular effects
of canthaxanthin (dose not specified) did not reveal any deposits in
the retina, but small alterations (prolongation) in dark adaptation
were observed. The significance of these results is controversial
(Hoffmann-La Roche, 1986).
In a 10-month experiment, three groups of chinchilla cross
rabbits were fed diets containing approximately 200 ppm ß-carotene,
canthaxanthin or ß-carotene+canthaxanthin. Total doses of ß-carotene
or of ß-carotene+canthaxanthin were approximately 11g; the total dose
of canthaxanthin was approximately 8 g. One of the canthaxanthin-
treated rabbits developed a paracentral chorioretinal defect that
increased under further treatment. While electroretinography in the
ß-carotene-treated animals showed slowly increasing scotopic a- and
b-wave peak latencies, the rabbits treated with canthaxanthin alone or
with ß-carotene showed hypernormal amplitudes at low cumulative doses
(ca. 1 g) and reduced amplitudes at higher doses (ca. 5 g) and peak
latencies increased remarkably. Histology and transmission electron
microscopy revealed a reduced retinal thickness in all carotenoid-
treated animals but in the canthaxanthin-treated rabbits there were
spotty degenerations and inclusions in the photoreceptor inner
segments. It was concluded that crystalline retinopathy may be a
specific effect in primates but that the functional retinal
alterations in humans that can be measured with the electroretinogram
are reproducible in the ERG of pigmented rabbits after canthaxanthin
treatment (Weber et al., 1987a; 1987b).
Groups of two chinchilla cross rabbits, average body weight
3.5 kg, were given intravenous liposome preparations daily for 19
days; one group received control liposomes yielding a daily dose of
13.7 mg of egg yolk phosphatidyl choline (PC), while the test group
received similar liposomes containing a daily dose of 2.1 mg
canthaxanthin. Two further groups of two rabbits were given the same
accumulated dose of 260 mg PC with or without 39.8 mg canthaxanthin,
but as a single injection. Electroretinography revealed that
canthaxanthin caused a depression of the a-waves and prolongation of
the scotopic a- and b-wave peak latencies. The single high-dose
injection of both control and canthaxanthin-containing liposomes
caused a transitory reduction in ERG aplitudes; the canthaxanthin-
containing liposomes also produced hypernormal a-waves within the
recovery time. Histologically, the canthaxanthin treated animals
showed alterations in the retinal pigment epithelium and
photoreceptors. It was concluded that an alteration in retinal
function and morphology occurs in the rabbit even after relatively low
doses of canthaxanthin (0.6 mg/kg bw/day for 19 days) (Weber et al.,
1987c).
2.2.7.2 Cats
Cats were fed up to 16 mg canthaxanthin/kg bw/day for 6 months.
No changes in ERGs were observed after 2 months. No crystals were
seen, but an orange sheen was reported to develop over the tapetum.
Light and transmission electron microscopy revealed fewer mature
pigment granules and more cytoplasmic vacuoles in the retinal pigment
epithelium. Electroretinograms performed at one and two months showed
no significant changes from baseline examinations (Scallon et al.,
1987, 1988).
2.3 Observations in man
Six out of a group of 42 patients with a history of urticaria
suffered a recurrence of their symptoms within 23 hours after an oral
challenge with 410 mg canthaxanthin taken as three divided doses over
3 hours (Juhlin, 1981).
The ingestion of doses of about 30-120 mg canthaxanthin daily
(approximately 0.4-1.7 mg/kg bw/day) for 3 months to several years in
medicinal or oral sun-tanning preparations has been associated with a
retinopathy in some individuals characterized by glistening, golden
crystals in the inner layers of the retina, up to 10-14 µm in size
(Boudreault et al., 1983; Cortin et al., 1984; Ros et al.,
1985). The crystalline deposits occur mainly in a ring between 5° and
10° around the fovea, less numerous in the fovea and rarely in the
foveola (Cortin et al., 1982). Occasionally, deposits have been
reported nasally of the disc (Metge et al., 1984) or scattered in
the posterior fundus (cited in Daicker et al., 1987) and in one case
only in the periphery of the fundus in the inner layer of a
retinoschisis (Cortin et al., 1982). A total dose of 75-178g
canthaxanthin has been found to cause this effect in 50% of subjects
and numerous cases have now been described (Franco et al., 1985;
Hennekes et al., 1985; McGuiness & Beaumont, 1985; Meyer et al.,
1985; Philipp, 1985; Saraux & Laroche, 1983; Weber et al., 1985b;
Weber & Goerz, 1986).
There are considerable differences among individuals in response
to canthaxanthin and there does not appear to be a clear relationship
between appearance of crystalline deposits and dose level or duration.
In one study, no crystals were detected in one subject after a total
intake estimated at 132 g canthaxanthin over a period of 7 years while
in another individual crystalline deposits were observed after an
accumulated total intake of 67.5 g in six years; crystal deposition
has even been described after exposure to 12-14 g canthaxanthin in 4
months. Canthaxanthin retinopathy was reported in 6 individuals who
claimed never to have taken canthaxanthin as a drug or sun-tanning
agent and the source of exposure to canthaxanthin in these cases in
unknown (Oosterhuis et al., 1988). Of 15 patients aged 9 to 72
years receiving a total accumulated dose of 10-170 g canthaxanthin
over a period of 1 to 10 years, six had crystalline retinal deposits
(Barker et al., 1987; Norris & Hawk, 1987). Conversely, in 23
patients receiving combined treatment with ß-carotene and
canthaxanthin for up to 2 years (average 2-6 months), no coloured
deposits were detected in the retina. However, from the data given in
this report it is not possible to determine the total doses involved
(Raab et al., 1985).
In a biostatistical evaluation of 253 cases having received
treatment with canthaxanthin, of whom 33 (15%) had retinal deposits,
the median yearly dose in subjects free from visible retinal deposits
was 5.3 g, whereas the corresponding figure for the group with pigment
deposits was 14.4 g. The lowest dose at which deposits were recorded
was 7 g canthaxanthin/year and no retinal deposits were found in
patients receiving less than 30 mg/day (Hoffmann-La Roche, 1986).
In a review of 259 reported cases with overall doses ranging from
3.6 g to 336 g over 3 months to 14 years, 92 patients showed crystals
but with clear dose/duration relationship (Barker, 1988). Maille
et al., (1988) reported that the dose associated with appearance of
crystal deposition in their patients varied from 7.92 g to 240 g but
that numerous subjects having ingested between 3.8 and 7.7 g did not
present with the retinopathy.
In view of the large differences reported in the doses of
canthaxanthin associated with crystal deposition in the retina, it has
been suggested that there are predisposing factors, including
co-administration of other carotenoids, age, intraocular hypertension
and particularly pre-existing retinal pigment epithelial defects
(Cortin et al., 1982, 1984; Maille et al., 1988; Metge et al.,
1984; Weber et al., 1985a; Oosterhuis et al., 1988, Pece et al.,
1988; Lonn, 1987).
In most cases, pigment deposition is not associated with any
detectable functional changes, but there have been occasional
complaints of dazzle or blurred vision (Cortin et al., 1984;
Hennekes et al., 1985; Philipp, 1985); visual field defects have
been described in only one report (Ros et al., 1985). The EOG is
normal or subnormal and dark adaptation may be delayed; scotopic
vision after exposure to glare may be reduced while the ERG is normal
or displays b-wave changes (Boudreault et al., 1983; Metge et al.,
1984; McGuiness & Beaumont, 1985; Weber et al., 1985b; Hennekes
et al., 1985; Philipp, 1985).
No adverse influence on visual function was reported after
treatment of 15 patients for up to 9 years (mean 4.9 years) with mean
total doses of 75.5 g (11-170 g) canthaxanthin, although 6 of these
patients displayed retinal crystals; dark adaptation was within normal
limits. EOG and PERG responses were not significantly different from
normal (Norris & Hawk, 1987). Similarly, 32 patients who had received
canthaxanthin therapy for 2-13 years (mean 5.8 years) did not have
visual symptoms, although 8 had retinal deposits. No abnormalities
related to crystal deposition could be found in visual field, dark
adaptation and EOG; there was no canthaxanthin-induced degeneration of
the retinal pigment epithelium (Nijman et al., 1986; Oosterhuis
et al., 1988).
In a study of 29 erythropoietic protoporphyria patients who had
received canthaxanthin for up to 10 years in daily doses of 30-150 mg
(total dose up to 170g canthaxanthin), dark adaptation and PERGs were
unaltered, but there was a slight decrease in the amplitude of the
scotopic b-wave in the ERG over the summer months. During the winter
this change was found to be reversible and, judged from this
parameter, it was reported that the no-effect-level appeared to be a
daily dose of 60 mg canthaxanthin (Schalch, 1988b).
Minor ERG changes (reduced b-wave amplitude) observed in EPP
patients, with or without crystalline deposits, did not cause symptoms
even after several years of therapy. The observations in this study
led the authors to conclude that the reduced b-wave is unlikely to
result from canthaxanthin acting as a filter reducing the number of
photons absorbed by photoreceptors as this would have caused
additional changes including increased latency and reduced amplitude
of the a-wave (Norris et al., 1987).
Evidence on the reversibility of retinal pigment accumulation is
controversial. Some workers have evaluated patients with retinal
crystals for up to 3 years after administration of canthaxanthin
ceased and reported no decrease in crystal deposits (Boudreault
et al., 1983; Weber et al., 1985a; Goerz & Weber, 1988; Weber &
Goerz, 1986; Lonn, 1987). Conversely, in a follow up over a mean
period of 47 months, retinal deposits in 7 out of 9 patients
decreased by 67±13% but no reduction was seen in the other two
patients (Malenfant et al., 1988). The number of retinal deposits
was evaluated in 9 patients, 2 to 4 times over a period of 55 months.
There was no significant difference observable after 9 months, but a
significant decrease in the number of retinal deposits was found after
26 months. The deposits disappeared slowly but some remained even
seven years after canthanxanthin was discontinued (Harnois et al.,
1988). Other workers have also noted some reversal of crystal
deposition after long recovery periods of up to 4 years (Oosterhuis
et al., 1988).
The eyes of a female patient, aged 72 years, who had retinal
deposits and who had died under anaesthesia, were examined by light
and electron microscopy, and the extracted pigment was examined by
mass and proton magnetic resonance spectroscopy. There were red,
birefringent crystals in the inner layers of the entire retina, which
were particularly large and numerous perifoveally where they were
clinically visible. The crystals were located in the inner neuropil,
where atrophy of the inner parts of the Müller cells was observed.
The compound was identical to canthaxanthin, and was present at up to
42 µg/g in the retina, along with a minor amount of other carotenoids.
Of the other ocular tissue, only the ciliary body contained measurable
amounts of canthaxanthin (Daicker et al., 1987).
Canthaxanthin was measured at autopsy in the tissues of 38
people, aged 22 to 96 years, none of whom were known to have received
canthaxanthin therapeutically or in sun-tanning preparations. The
tissues examined were mesenteric and sub-cutaneous fat, skin, liver,
spleen and blood serum. The highest concentrations were found in
omentum and sub-cutaneous fat (mean concentrations 0.2 and 0.3 µg/g
respectively). The mean concentrations in other tissues were: liver,
0.08 µg/g; skin and spleen, 0.04 µg/g; and serum, 0.024 µg/ml
(Hoffmann-La Roche, 1986).
Fat samples from mesenterium and omentum and a liver sample were
taken at autopsy from a 71-year old woman who had died from bronchial
carcinoma. The patient had previously ingested approximately 45 mg
canthaxanthin/day for four years (total dose approximately 65 g) in a
sun-tanning preparation. The concentrations of canthaxanthin in
omentum and mesenteric fat were 270 µg/g and 158 µg/g, respectively.
Lower levels of 5 µg/g were found in the liver (Hoffmann-La Roche,
1986).
Biopsy samples of orange-coloured fat (omentum) were obtained
from two patients undergoing surgery. In one case, the woman had
taken a total dose of about 6g canthaxanthin in a tanning preparation
during 1983/84 and had stopped this intake 1-1´ years before the
biopsy; fat and serum canthaxanthin levels were 49 µg/g and 69 µg/1,
respectively. In the second case, the patient had taken a total dose
of approximately 16g over 2´-3 years and the concentration in omentum
was 34 µg/g (Hoffmann-La Roche, 1986).
3. COMMENTS AND EVALUATION
In reviewing the results of two long-term/carcinogenicity studies
in mice and rats, the Committee noted that these did not provide
evidence of carcinogenicity but, at high dose levels, canthaxanthin
produced liver damage in the rat (with a non-dose-related increased
incidence of benign nodules in female rats); the mouse appeared less
sensitive to hepatic injury. It was concluded that, in addition to
the eye, the liver was a target organ for canthaxanthin.
In the long/term studies in rats, it was not possible to
establish a no-effect level, but the Committee was informed that
another long/term study in rats was in progress aimed at establishing
a no-effect level with respect to hepatic pathology.
On the basis of distribution studies using radiolabelled
canthaxanthin, relatively high concentrations accumulated in the eye
in all mammalian species studied. However, to date, crystal
deposition has been observed only in the human retina. Therefore, the
animal species studied did not provide a suitable model for the study
of the pathogenesis and reversibility of this phenomenon. However,
changes in ERGs in humans were reproduced in the ERGs of pigmented
rabbits.
Although the Committee concluded that the long/term toxicity in
rats raised questions of potential hepatotoxicity, these may be
answered by obtaining clinical data derived from human subjects
showing retinal pigment deposition. However, the Committee considered
that the primary problem related to canthaxanthin is its crystal
deposition in the human retina.
In view of the irreversibility or very slow reversibility of the
retinal crystal deposition, the significance of which is not known,
the Committee was unable to establish an ADI for canthaxanthin when
used as a food additive or animal feed additive. Therefore, the
previous temporary ADI was not extended.
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