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    CITRANAXANTHIN

    EXPLANATION

         Citranaxanthin is a synthetic compound. Its major present-day use
    is as an animal feed additive to impart a yellow colour to chicken fat
    and egg yolks. It can be used as colouring matter in the same manner
    and for the same purposes as other carotenoids (beta-carotene,
    beta-apo-8'-carotenal, beta-apo-8'-carotenoic acid ethyl ester, and
    canthaxanthin).

         Citranaxanthin has about two-thirds of the vitamin A activity of
    beta-carotene in chickens. This is equivalent to 1,100 I.U. of vitamin
    A per 1 mg of citranaxanthin.

    BIOLOGICAL DATA

    Biochemical aspects

    Absorption, distribution, and excretion

         Twenty female Sprague-Dawley rats weighing about 170 g each
    received single i.v. doses of 14C-citranaxanthin dissolved in
    polyethylene glycol. Each dose was equivalent to 1 mg/kg b.w. At 1,
    24, 72, and 168 hours after administration five of the rats were
    sacrificed. The 14C content of the following organs and tissues were
    determined: heart, liver, lungs, spleen, eyes, pituitary, brain,
    kidneys, pancreas, stomach, duodenum, thymus, periovarian and
    perirenal fats, ovaries, skin, hair, adrenals, thyroid, and muscle.
    The absolute amounts of radioactivity found in each organ were
    particularly high in the liver, lungs, and spleen. The radioactivity
    found in these organs ranged from 49% (1 hour) to 15% (168 hours) of
    the dose in the liver, 14% (1 hour) to 2.7% (168 hours) of the dose in
    the lungs, and 2.2% (1 hour) to 0.85% (168 hours) of the dose in the
    spleen.

         The doses recovered in other organs after 1 hour were 0.7% in the
    heart, 0.1% in the brain, and 0.6% in the kidneys. Muscle and adipose
    tissue contained 0.3 and 2.6%, respectively of the dose administered
    after 1 hour, thus emphasizing the hydrophobic character of
    citranaxanthin. All other organs examined contained levels of
    radioactivity less than 0.1% of the dose/g 1 hour after
    administration. These levels, which were very low and were associated
    with large errors of measurement, decreased even further over the 168
    hours after administration. The changes in levels of radioactivity
    with time were similar in all organs and tissues, decreasing slowly
    overall. A half-life of about 140 hours for this decrease was
    estimated by a logarithmic regression method (Morgenthaler, 1979).

         Four female Sprague-Dawley rats weighing about 160 g each
    received 14C- and 3H-citranaxanthin orally by gastric tube.
    Citranaxanthin was administered as an oily suspension in
    carboxymethyl-cellulose. In both cases 95-98% of the radioactivity was
    recovered in faeces (69-80% (first day); 15-28% (second day); 0.7%
    (third day)). After 8 days, 0.2-0.8% of the radioactivity was found in
    the liver, 0.2% in the urine, and 0.5% in the carcass
    (Morgenthaler, 1978).

         The average absorption rate of citranaxanthin in poultry was 50%.
    The rest was excreted in the faeces. About two-thirds of the absorbed
    amount was metabolized to vitamin A; about 40% was present in blood
    and other organs. One-third of the absorbed citranaxanthin was
    deposited in the fat of various tissues, in skin, and in egg yolk
    (Crina, 1974a,b).

    Vitamin A activity of citranaxanthin

         One hundred and twelve chickens (one-day old) were fed a diet
    free of vitamin A for 3 weeks. Divided groups received doses of 1.2,
    2.4, 4.8, 6.0, 12.0, or 24.0 mg citranaxanthin daily over a period of
    five days. Liver analyses showed storage of citranaxanthin and vitamin
    A derived from citranaxanthin (Tiews, 1968a).

         Chicks and quails received 40 ppm citranaxanthin and
    beta-carotene over 2 weeks in their diets after a 3-week rearing
    period without vitamin A. The vitamin A activity of citranaxanthin in
    chickens was higher than that of beta-carotene; in quails it was lower
    (Tiews, 1968b).

         After a 21-day vitamin A-depletion period, groups of 20 laying
    hens were fed diets containing 1.5, 3, 6, or 10 ppm citranaxanthin.
    Colouration of egg yolk by citranaxanthin was observed. This effect
    was found to be influenced by dose and particle size (Kolk, 1974).

         After a vitamin A-depletion period of 3 weeks, groups of 14 or 20
    chickens were fed diets containing 8.6, 14, 24, or 40 ppm
    citranaxanthin for 2 weeks. The control groups received feed which
    contained the same amounts of beta-carotene instead of citranaxanthin.
    Determination of the liver vitamin A content showed that the formation
    of vitamin A from citranaxanthin depended upon the crystal size. The
    relative vitamin A activity was 66 to 94% of the activity compared
    with beta-carotene (Kolk, 1974).

         The vitamin A activity of citranaxanthin was tested in chicken
    liver storage tests (Table 1). From this test and others it can be
    concluded that citranaxanthin has about two-thirds of the vitamin A
    activity of beta-carotene. This is equivalent to 1,100 I.U. of vitamin
    A/mg citranaxanthin (Kolk, 1974).

    Toxicological studies

    Special study on inhalation

    Rats

         Twelve animals were exposed to citranaxanthin crystalline/ air
    mixture at a concentration of 0.29 mg/liter of air during 8 hours. No
    deaths occurred. Toxic symptoms were not evident; only slight mucous
    membrane irritation was observed (BASF, 1972a).

    Special study on mucous membrane irritation

    Rabbits

         Fifty mg citranaxanthin crystalline was applied to the eyes of
    rabbits. No irritation was seen. After 1 hour the eyes had a red
    appearance; after 24 hours and 8 days these symptoms were not
    noticeable (BASF, 1972a).

    Table 1.  Vitamin A activity of citranaxanthin
                                                                        

    Dosage         Vitamin A      Vitamin A           Relative vitamin A
    (ppm in feed)  (I.U./liver)   (I.U./mg ingested   activity
                                  carotenoid)         (ß-carotene = 100%)
                                                                        

    ß-Carotene "water soluble"

     8.65            900          195                 100
    14.4           1,803          222                 100
    24.0           2,596          191                 100

    Citranaxanthin

     8.65            544          118                  60
    14.4           1,080          124                  56
    24.0           1,991          144                  76
                                                                        

    Special study on reproduction

    Rats

         A 3-generation reproduction study with groups of 20 male and 20
    female Sprague-Dawley rats was conducted in which the animals were
    administered 1000, 3300, or 10,000 ppm citranaxanthin (8.6% dry
    powder, equivalent to 86, 284, and 860 ppm active ingredient). Another
    group was administered 9000 ppm dry powder without active ingredient.
    A control group remained untreated. Treatment of both males and
    females started 7 weeks before breeding and continued during the
    mating, pregnancy, and rearing periods. Fertility and reproduction
    performance were not influenced in any generations (F0, F1, or
    F2) or groups. Mating, pregnancy, litter size, birth weights, and
    rearing were within normal limits. The indices of fertility,
    pregnancy, viability, and lactation did not differ between control and
    treatment groups. Animals of the F2-generation were investigated for
    malformations; none were observed. Behaviour, appearance, feed and
    water intake, and weight gain were not affected by treatment in any
    generations or groups. No macroscopic pathological effects were
    observed in parents or their offspring. The final macroscopic
    examination of the F2-generation animals after 9 weeks of age
    revealed no changes due to treatment. Comparisons of organ weights and
    histological examinations showed no differences among groups
    (Leuschner et al., 1976a).

    Special study on skin irritation

         Crystalline citranaxanthin was applied to the skin of rabbits in
    a 50% aqueous suspension for 20 hours. A red-brownish colouring was
    observed, but inflammation was not detected (BASF, 1972a).

    Acute toxicity
                                                                        

    Species        Route     LD501             Reference
                             (mg/kg b.w.)
                                                                        

    Mouse (10)     i.p.      > 6,400           BASF 1972a
    Rat (10)       oral      > 6,400           BASF 1972a,b
    Dog (6)        oral      > 1,590           Leuschner, 1976c
                                                                        

    1    Higher doses could not be administered in these studies for
         technical reasons.

    Rats

         Citranaxanthin was administered in the diet to groups of 13 to 15
    male and female Sprague-Dawley rats at dose levels of 10, 20, 50, or
    100 mg/kg/day for one month. Growth rates and feed consumption were
    not significantly different between the treated and the control groups
    during the test period. No toxic symptoms or abnormalities in
    urinalysis, biochemical values, blood analysis, wet organ weights, or
    histopathological changes were found (Kawase et al., 1972).

         Four groups of 20 or 30 male and female Sprague-Dawley rats were
    maintained over a period of 91 days on a diet containing 25,000,
    50,000, or 100,000 ppm 10% citranaxanthin dry powder (equivalent to
    2,500, 5,000, and 10,000 ppm active ingredient). Citranaxanthin was
    tolerated without externally recognizable toxic symptoms and without
    impairment of feed ingestion or growth over a period of 91 days. A
    significant increase in total serum lipids was observed in both males
    and females during the treatment period. GPT values were temporarily
    increased after 8 weeks, but not after 12 weeks.

         The absolute and relative weights of the liver and kidneys showed
    a significant increase when compared with those of the control group.
    All the increases were reversible in the post-observation period,
    except that the increase in kidney weight did not revert completely.

         No salient pathological changes were observed in any goups. A few
    cases of enlargement of the liver proved after histological
    investigation to be hyperaemia, in some cases diffuse and in other
    cases patchy. Accumulated changes in the cylinders containing protein
    in the distal tubule sections of the kidneys were found in all groups
    (Hempel et al., 1973).

    Long-term studies

    Rats

         Groups of 25 male and 25 female Sprague-Dawley rats were fed for
    two years with graded levels of 1000, 3300, or 10,000 ppm 8.6%
    citranaxanthin dry powder (equivalent to 86, 284, and 860 ppm active
    ingredient). Another group was administered 9000 ppm dry powder
    without active ingredient. A control group remained untreated. After 6
    months the highest concentration of 10,000 ppm was raised to
    20,000 ppm (1720 ppm active ingredient) and the concentration of the
    placebo was raised to 18,000 ppm for the remaining period of the
    experiment.

         There were no signs of incompatibility in any groups throughout
    the experiment. Appearance, behaviour, weight gain, haematology,
    clinical chemistry, urinalysis, weight of organs, and their autopsy
    did not reveal any influence of treatment. There were no histological
    changes in organs or tissues. The number and kind of tumours or
    mortality did not differ among groups (Leuschner et al., 1976b).

    Dogs

         A 180-day feeding trial with 5 groups of 8 beagle dogs (4 males
    and 4 females) was performed with graded levels of 1000, 3300, or
    10,000 ppm 8.6% citranaxanthin dry powder (equivalent to 86, 284, and
    860 ppm active ingredient). Another group was administered dry powder
    without active ingredient. A control group remained untreated. In all
    groups with citranaxanthin the faeces had a remarkable red-brown
    colour. At the highest level the faeces were more liquid than normal.
    Feed intake decreased in a dose-dependent manner due to palatability
    problems. The dry powder group without citranaxanthin also had
    decreased feed intake.

         Appearance, behaviour, weight gain, haematology, clinical
    chemistry, electrocardiography, urinalysis, weight of organs, and
    their autopsy did not reveal any influence of treatment. There were no
    pathological or histological changes in organs or tissues
    (Leuschner et al., 1975).

    Observations in man

         No information available.

    COMMENTS AND EVALUATION

         The major present-day use of citranaxanthin is as an animal feed
    additive to impart a yellow colour to chicken fat and egg yolk. It may
    also be used as a colouring agent by adding it directly to food.

         If the substance were to be used as a direct food colouring
    agent, the data were not sufficiently comprehensive for evaluation
    (e.g. only one lifetime feeding study was available). The Committee
    concluded that further data of the type outlined in Annex III of
    "Principles for the Safety Assessement of Food Additives and
    Contaminants in Food" for synthetic food colours are required before
    the substance can be fully evaluated for direct food use (Annex 1,
    reference 76).

         For its use as an animal feed additive, an evaluation could not
    be made because the data base did not include sufficient information
    on the nature of residues to be found in animal-derived foodstuffs and
    because there was no information concerning the use levels that would
    constitute good animal husbandry practice.

    REFERENCES

    BASF (1972a). Ergebnisse der gewerbetoxikologischen Vorprufung.
    Unpublished report from BASF Department of Toxicology. Submitted to
    WHO by BASF Aktiengesellschaft, Ludwigshafen, FRG.

    BASF (1972b). Report on tests for acute oral toxicity of
    citranaxanthin dry powder in rats. Unpublished report from BASF
    Department of Toxicology. Submitted to WHO by BASF Aktiengesellschaft,
    Ludwigshafen, FRG.

    Crina (1974a). Citranaxanthin for broilers No. 1. Unpublished report.
    Submitted to WHO by BASF Aktiengesellschaft, Ludwigshafen, FRG.

    Crina (1974b). Citranaxanthin for broilers No. 2. Unpublished report.
    Submitted to WHO by BASF Aktiengesellschaft, Ludwigshafen, FRG.

    Hempel, K.J., Zeller, H., Kirsch, P., Koob, K., & Freisberg, K.O.
    (1973). Report on the examination of citranaxanthin 10% dry powder in
    a 91-day feeding test with rats. Unpublished report from Allgemeines
    Krankenhaus, Pathological Institute of Heidberg, Hamburg and BASF,
    Department of Toxicology. Submitted to WHO by BASF Aktiengesellschaft,
    Ludwigshafen, FRG.

    Kawase, S., Komatsu, Y., Suzuki, Y., Nishida, S., & Kobayashi, A.
    (1972). Subacute toxicity of citranaxanthin. J. Med. Soc. Toho.,
    Japan, 19, 499-504.

    Kolk, J.H.H. (1974). About vitamin A efficiency and pigmenting effect
    of three citranaxanthin preparations at chicks and quails with
    different crystal size. Dissertation, University of Munich.

    Leuschner, F., Leuschner, A., Schwerdtfeger, W., & Dontenwill, W.
    (1975). Oral toxicity of citranaxanthin, batch BASF No. 10 - called
    for short "cn" - and of the substance without agent - called for short
    "swa" - in the beagle dog (repeated dosage over 6 months). Unpublished
    report from Laboratorium für Pharmakologie und Toxikologie, Hamburg,
    to BASF. Submitted to WHO by BASF Aktiengesellschaft,
    Ludwigshafen, FRG.

    Leuschner, F., Leuscher, F., & Dontenwill, W. (1976a). Oral toxicity
    of citranaxanthin-trockenpulver, batch BASF No. 10 - called for short
    "cn" - and of the substance without agent, batch 908 E 953 called
    "swa" - in the Sprague-Dawley rat (reproduction study covering three
    succeeding generations). Unpublished report from Laboratorium fur
    Pharmakologie und Toxikologie, Hamburg, to BASF. Submitted to WHO by
    BASF Aktiengesellschaft, Ludwigshafen, FRG.

    Leuschner, F., Leuschner, A., Schwerdtfeger, W., & Dontenwill, W.
    (1976b). Two-year toxicity testing of citranaxanthin dry powder, batch
    BASF No. 10 - called for short "cn" - and of the substance without
    agent, batch 908 E 953 called "swa" - in the Sprague-Dawley rat.
    Unpublished report from Laboratorium fur Pharmakologie und
    Toxikologie, Hamburg, to BASF. Submitted to WHO by BASF
    Aktiengesellschaft, Ludwigshafen, FRG.

    Leuschner, F. (1976c).  Prufung der akuten Toxizität yon
    citranaxanthin. Charge BASF NR. 10- kurz "CN" genannt- an
    mischrasaigen Hunden bei peroraler Verabreichung. Unpublished report
    from Laboratorium fur Pharmakologie und Toxikologie, Hamburg, to BASF.
    Submitted to WHO by BASF Aktiengesellschaft, Ludwigshafen, FRG.

    Morgenthaler, H. (1978). Citranaxanthin balance and excretion study.
    Unpublished report from BASF Pharma Division. Submitted to WHO by BASF
    Aktiengesellschaft, Ludwigshafen, FRG.

    Morgenthaler, H. (1979). Organ citranaxanthin - distribution.
    Unpublished report from BASF Pharma Division. Submitted to WHO by BASF
    Aktiengesellschaft, Ludwigshafen, FRG.

    Tiews, J. (1968a). Certified report about vitamin A activity of
    citranaxanthin and other carotenoids. Unpublished report. Submitted to
    WHO by BASF Aktiengesellschaft, Ludwigshafen, FRG.

    Tiews, J. (1968b). Citranaxanthin for egg yolk pigmentation.
    Proceedings from Lohmann Nutrition Conference, Cuxhaven, pp. 47-50.
    


    See Also:
       Toxicological Abbreviations
       CITRANAXANTHIN (JECFA Evaluation)