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. 5. REFERENCES ARDEN, G.B., OLUWOLE, J.O., POLKINGHORNE, P., BIRD, A.C., BARKER, F.M., NORRIS, P.G. & HAWK, J.L. (1989). Monitoring of patients taking canthaxanthin and carotene: an electroretinographic and ophthalmological survey. Human Toxicol., 8: 439-450. ASTORG, P.O., GRADELET, S., LECLERC, J., CANIVENC, M.C., & SIESS, M.H. (1994). Effects of B-carotene and canthaxanthin on liver xenobiotic-metabolizing enzymes in the rat. Fd Chem. Toxicol., 32: 735-742. BARKER, F.M. & FOX, J.G. (1992). The ferret ERG in high dosage canthaxanthin administration. Unpublished research report submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. BAUSCH, J. (1992a). (14C)-Canthaxanthin: Absorption, Distribution and Excretion Following Oral Administration at Steady State in the Cynomolgus Monkey. Unpublished research report No. 106 798, submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. BAUSCH, J. (1992b). Canthaxanthin Metabolic Studies: Comparison of Rat Results with Monkey Results. Unpublished research report No. B-106 799, submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. BAUSCH, J., GLATZLE, D., BRUCHLEN, M., & RINGENBACH (1991). Canthaxanthin distribution studies in pigmented rats. Unpublished research report No. B-106 772 submitted to WHO by F. Hoffmann-La Roche & Co.. Basel, Switzerland. BRUININK, A., COHN, W., & WEISER, H. (1992). Chick embryonic neuronal retina, RPE, brain and meninges cell cultures as a model for canthaxanthin induced alterations in the eye. Unpublished research report submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. BUSER, S. (1992a) Canthaxanthin (Ro 01-9915) in a long-term study with male rats (feed admixture). Unpublished research report B-157'342, submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. BUSER, S. (1992b). Canthaxanthin (Ro 01-9915) in a long-term study with female rats (feed admixture). Unpublished research report B-157'343 submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. BUSER, S., GORALCZYK, R., BAUSCH, J., SCHÜEP, W. (1993). Canthaxanthin (Ro 01-9915) in a long-term study with Cynomolgus monkeys (oral gavage); 3-year interim report. Unpublished research report no. B-161'152 submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. BUSER, S. (1994). Canthaxanthin. A critical approach on the experimental procedures and results in two subsequent long-term studies with rats. Reports nr. 119'970 of March 17, 1988; nr. 157'342 of August 31, 1992; nr. 157'343 of August 31, 1992. Unpublished report submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. BUSER, S., GORALCZYK, R., BAUSCH, J., SCHÜEP, W. (1994). Canthaxanthin (RO 01- 9915) in a long-term study with Cynomolgus monkeys (oral gavage); 3-year final report. Unpublished research report no. B-161'152 submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. EL-ATTAR, T.M. & LIN, H.S. (1991). Effect of retinoids and carotenoids on prostaglandin formation by oral squamous carcinoma cells. Prostaglandins Leukotrines and Essential Fatty Acids, 43: 175-178. FOX, J.G., TAYLOR, N.S., & BLANCO, M. (1992). Is the Ferret a Model to Study Canthaxanthin Retinopathy? Report submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. GELEICK, H, & KLECAK, G. (1983). Determination of the sensitizing potential of canthaxanthin crystalline in guinea pigs by the Optimization Test (Maurer). Unpublished research report no. 104'987 submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. GENSLER, H.L. (1989). Reduction of immunosuppression in UV-irradiated mice by dietary retinyl palmitate plus canthaxanthin. Carcinogenesis, 10: 203-207. GLATZLE, D., & BAUSCH, J. (1989). Canthaxanthin Balance Studies. Unpublished research report No. B-106 714 submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. GORALCZYK, R. (1993). Post mortem examination of ferret eye tissue after long term feeding of canthaxanthin. Unpublished research report No. B-158'296 submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. GORALCZYK, R., BAUSCH, J. & WEISER, H. (1993). Dose-dependent occurrence of canthaxanthin-related birefringent particles in the retina of broiler chicks. Unpublished research report No. B-158'295 submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. GORALCZYK, R. & WEISER, H. (1992). Birefringent particles in eyes of chicks after a very high dietary intake of canthaxanthin. Unpublished research report B-158'278 submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. HANDELMAN, G.J., DRATZ, E.A., REAY, C.C. & VAN KUIJK, J.G. (1988). Carotenoids in the human macula and whole retina. Invest. Ophthalmol. Vis. Sci., 29: 850-855. HANDELMAN, G.J., SNODDERLY, D.M., & KRINSKY, N.I. (1991). Biological control of primate macular pigment. Invest. Ophthalmol. Vis. Sci., 32: 257-267. HARNOIS, C., CORTIN, P., SAMSON, J., BOUDREAULT, G., MALENFANT, M. & ROUSSEAU, A. (1988). Static perimetry in canthaxanthin maculopathy. Arch. Ophthalmol., 106: 58-60. HARNOIS, C., KUWABARA, T., BOUDREAULT, G., MALENFANT, M. AND ROUSSEAU, A. (1990). Canthaxanthin retinopathy in monkeys: clinical and histopathological studies. Unpublished research report submitted to WHO by F. Hoffman-La Roche & Co., Basel, Switzerland. KÖPCKE, W, BARKER, F.M., & SCHALCH, W. (1994). Canthaxanthin- deposition in the retina. A biostatistical evaluation of 411 cases taking this carotenoid for medical or cosmetical purposes. Unpublished research report submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. LEYON, H., ROS, A.M., NYBERG, S. & ALGVERE, P. (1990). Reversibility of canthaxanthin deposits within the retina. Acta Ophthalmol., 68: 607-611. NORRIS, P. G., & HAWK, J. L. M. (1990). A study of hepatic function during carotenoid therapy for erythropoietic protoporphyria. Unpublished report submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland. RYBSKI, J.A., GROGAN, T.M., AICKIN, M. & GENSLER, H. (1991). Reduction of murine cutaneous UVB-induced tumour-infiltrating T lymphocytes by dietary canthaxanthin. J. Invest. Dermatol., 97: 892-897. SCHIEDT, K., BISCHOF, S. & GLINZ, E. (1991). Recent progress on carotenoid metabolism in animals. Pure App. Chem., 63: 89-100. SCHIEDT, K., BISCHOF, S., & GLINZ, E (1992). Analysis of carotenoids and retinoids in eye tissues of various species after canthaxanthin long-term treatment. Unpublished research report no. B-157'006 submitted to WHO by F. Hoffmann-La Roche & Co., Basel, Switzerland.
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)