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    CAROTENES FROM NATURAL SOURCES (ALGAL AND VEGETABLE)

    First draft prepared by
    Dr G.J.A. Speijers
    National Institute of Public Health
    and Environmental Protection
    Laboratory for Toxicology, Bilthoven, The Netherlands

    1.  EXPLANATION

         Carotenes from natural sources were reviewed at the eighteenth,
    thirty-first and thirty-fifth meetings of the Committee (Annex 1,
    references 35, 77 and 88).  At its thirty-first meeting, the
    Committee noted that, while there was a substantial toxicological
    database relating to carotenes and an ADI had been established for
    synthetic -carotene, the same ADI was not applicable to natural
    carotenes as they did not comply with the specifications for
    -carotene.

         At the thirty-fifth meeting, the Committee concluded that there
    was insufficient evidence to indicate that data relating to one
    species of  Dunaliella algae could be applied to others and that
    the specifications of the test materials were so different from one
    another that the results of the toxicity tests could not be
    generalized.  There were insufficient data to evaluate any of these
    materials for the purpose of establishing an ADI.  The Committee
    concluded that carotene isolated from algal sources would be
    acceptable for food additive use if it was of sufficient purity to
    meet the specifications for synthetic -carotene.  Acceptance of
    algal biomass or crude extracts of carotene from algal sources for
    use as food additives would be contingent on the provision of
    evidence of the safety of such materials.

         At its thirty-fifth meeting, the Committee considered limited
    short-term toxicological studies on material stated to have been
    prepared from three different algal species designated  Dunaliella
     bardawil, D. salina and D. kona (Annex 1, reference 88).  At its
    present meeting, the Committee was informed that  Dunaliella
     bardawil, D. kona and D. salina were identical, and that,
    according to current nomenclature, the species used commercially was
     Dunaliella salina.  Some of the preparations produced from this
    species were dehydrated powders prepared by lyophilization or spray-
    drying and others were vegetable oil extracts.1


                 

    1  Although the Committee acccepts that this report does not cover
         different  Dunaliella species, the studies are summarized with
         original names mentioned in the reports or publications
         submitted.

         With respect to the carotene preparations derived from
    extraction of vegetables, mainly carrots, alfalfa or vegetable oil,
    the Committee at the thirty-first meeting (Annex 1, reference 77)
    felt that the need for toxicity tests may be obviated if detailed
    analytical data were supplied to confirm that natural toxicants
    occurring at low levels in food/feed stuffsare not concentrated in
    the extract and that levels of use would not materially exceed the
    levels of exposure that would result from normal use.

         As no toxicological monograph has been prepared previously by
    the Committee on carotenes from natural sources, data that have been
    reviewed at previous meetings are incorporated in the present
    monograph along with the new data that have become available. 
    Because this monograph covers the data on both algal and vegetable
    carotene preparations, a modified form of the general monograph
    format has been used, summarizing in order biological data on spray
    dried concentrated, lyophilized or dehydrated preparations of
     Dunaliella, then data on vegetable oil extracts of  Dunaliella,
    then data on carotene extracts from carrots, grass alfalfa and
    vegetable oil.

    ALGAL CAROTENE PREPARATIONS
    SPRAY DRIED CONCENTRATED, LYOPHILIZED OR DEHYDRATED
    PREPARATIONS OF  DUNALIELLA SALINA
    (syn  DUNALIELLA BARDAWIL and DUNALIELLA KONA)

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion.

         Male weanling CD rats were fed  ad libitum a retinol-deficient
    diet.  After 60 days, the retinol content of the livers was 4-5 g. 
    Depleted rats were allocated to 9 groups of 6 animals, housed
    individually, and fed a retinol-deficient diet supplemented as
    follows: Group 1, retinol at 7.5 mg/kg diet; Groups 2-4, all- trans
    -carotene at 12, 29 or 48 mg/kg diet respectively; Groups 5-7,
    lyophilized  Dunaliella contributing 29, 58 or 112 mg -carotene/kg
    diet respectively; Group 8, maize oil extract of  Dunaliella
    providing 16 mg -carotene/kg diet; and Group 9, no supplementation. 
    After seven days repletion, livers were taken for analysis for
    retinol, retinol isomers and -carotene.  The liver analyses
    revealed a comparable content of retinol related to dose of
    carotene, irrespective of source whether synthetic, algal biomass or
    algal oil extract.  Rats fed the algae or the algal extract-
    supplemented diets accumulated 9- cis retinol in addition to the
    all- trans isomer.  Rats fed synthetic -carotene, lyophilized
    algae or algal oil had a liver retinol:-carotene ratio of about
    3:1.  Rats fed algae or algal oil accumulated 9- cis -carotene and
    all- trans -carotene in the liver in a ratio similar to that
    present in the algae.  It was concluded that dried  Dunaliella
     bardawil or an oil extract of the alga can serve as a dietary
    natural -carotene source which can satisfy the total requirement of
    retinol in rats (Ben-Amotz  et al. 1988).  [Note: this study is
    again cited below in discussion of absorption of oil-extracted algal
    carotene]

         A 45-day-feeding study with 21-day old Sprague-Dawley rats was
    performed to compare the bioavailability of four sources of
    -carotene; Spray dried  Dunaliella salina (0.61 % -carotene),
    -carotene oil extract of  Dunaliella salina (24.83 % -carotene),
    oleoresin of carrots (11.5 % -carotene) and synthetic -carotene
    (100 %).  The diet with  Dunaliella salina powder (59 g/kg diet)
    was corrected for the amount of sucrose.  The diets were made equal
    in the percentage of -carotene, and the control diet contained
    0.036 % -carotene.

         The rats on the  Dunaliella salina diets grew more rapidly. 
    Gross macroscopy at autopsy revealed no alterations in the rats.

         The bioavailability of  Dunaliella salina as a source of
    -carotene and oil extract of -carotene from  Dunaliella salina
    was higher than that of -carotene from oleoresin of carrots or
    synthetic -carotene.  These results may be due to the presence of
    the extra lipids in both the dried  Dunaliella salina which
    contains 8.6 % lipid and the oil extract of  Dunaliella salina. 
    These results lead to the recommendation that dried  Dunaliella
     salina, when used as a source of -carotene, should be consumed in
    oil to increase -carotene bioavailability.  This is because
    activity of carotene dioxygenase, the enzyme responsible for the
    conversion of -carotene to vitamin A, is increased in the presence
    of oil (Ghazi  et al., 1992).

         In one experiment, groups of 1-day-old white Leghorn chicks
    received a retinol-deficient semi-purified diet or a similar diet
    supplemented with 8.04 mg retinol/kg diet, or 30 mg synthetic
    -carotene/kg diet or lyophilized  D. bardawil at 1 g algae/kg
    diet.  The algal powder contained 30 g -carotene and 200 g NaCl/kg
    and provided 30 mg -carotene/kg diet.  In a second experiment, four
    similar groups were used except that the lyophilized algal powder
    was replaced with a similar concentration of drum-dried algae.  The
    drum-dried algal powder contained 34 g -carotene, 260 g NaCl, 180 g
    glycerol, 5.5 g chlorophyll, 210 g protein, 170 g carbohydrate and
    120 g lipid/kg (34 mg -carotene/kg diet).  A third experiment
    utilized three groups of 15 one-day-old chicks which received
    retinol-deficient diet alone, or supplemented with lyophilized
     D. bardawil at 0.58 g/kg diet, or with drum dried algae at 1 g/kg
    diet.  In each experiment, the chicks were assessed visually and
    weighed daily for 5 weeks and at termination serum and liver were
    analysed for retinol, -carotene and lutein.

         After an initial lag, the chicks grew equally well on diets
    containing retinol, -carotene or algae in all experiments.  Serum
    and liver concentrations of retinol were normal in all cases except
    for the chicks receiving retinol-deficient diets without
    supplements.  The serum of chicks fed the algal-supplemented diets
    contained lutein but no -carotene although the ratio of -carotene
    to lutein in the algae exceeded 15:1.

         In a separate experiment, two groups of 3 egg-laying hens
    received a control diet containing 150 g maize meal/kg or the same
    diet supplemented with 4 g lyophilized  D. bardawil/kg.  The algal
    preparation contained 50 g -carotene and 300 g NaCl/kg (200 mg
    -carotene/kg diet).  Eggs from these hens showed an enhanced yolk
    colour attributable to lutein; no -carotene was present in the egg
    yolk (Ben-Amotz 1986).

    2.1.2  Biotransformation

         No information available.

    2.1.3  Effects on enzymes and other biochemical parameters

         No information available.

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

    2.2.1.1  Mice

         Acute toxicity of  Dunaliella bardawil spray dried powder was
    established in an LD50 test with mice.  A single dose of 2.5, 5.0
    or 10.0 g  Dunaliella bardawil/kg diluted with CMC-Na solution was
    administered by oral intubation.  The observation period was 14
    days, and mortality, general symptoms, body weights and gross
    necropsy examination were recorded.  The LD50 value was greater
    than 10 g  Dunaliella bardawil/kg for both male and female mice
    (Aruga, 1987).

    2.2.1.2  Rats

         Male Sprague-Dawley rats were given dried  Dunaliella by
    gavage at a dose of 5 g/kg bw  and observed for the subsequent 14
    days.  There were neither mortalities nor overt signs of toxicity
    and all the rats gained weight during the observation period (Lock
    1985).

    2.2.2  Short-term toxicity studies

    2.2.2.1  Mice

         In the search for an antioxidative-anticarcinogenic substance,
    the effects of repeated ingestion (no dose level given) of spray
    dried  Dunaliella bardawil on mammary growth and endocrine
    parameters were examined in mice.  In an additional group the mice
    received also the vitamin A-deficient synthetic standard diet
    supplemented with synthetic all-trans -carotene, whereas the
    control animals received the normal synthetic diet adequate in
    vitamin A.  The concentration of -carotene in both test diets was
    0.55 mg/kg.  The ingestion of the  Dunaliella bardawil-containing
    diet between 20 and 120 days of age showed no deleterious side-
    effects on mammary gland and uterine growth nor mammatrophic hormone
    secretion, these results were similar to previously-observed results
    in aged and mammary tumour-bearing mice.  Puberty and body growth
    were accelerated by  Dunaliella bardawil compared to the synthetic
    all-trans -carotene (Nagasawa  et al., 1989).

    2.2.2.2  Rats

         Two groups of 10 male and 10 female weanling Sprague-Dawley
    rats, caged individually, were given powdered diets containing 0 or
    10% algal -carotene powder for 12 weeks.  Body weight gain and food
    intake were recorded at intervals up to and at termination when the
    animals were autopsied.  Weights of heart, lungs, liver, kidneys,
    spleen, gonads and adrenals were determined at autopsy and blood was
    collected for determination of serum glucose, ASAT, ALAT, alkaline
    phosphatase, uric acid, BUN, triglycerides and cholesterol.  One
    male rat in the treated group died during the study from
    "non-specific problems" not related to treatment.

         Significant differences were observed in neither food intake
    nor body weight gain between treated and untreated animals of either
    sex and there were no treatment-related differences in organ
    weights.  Except for one treated male which displayed elevated ASAT
    and ALAT levels, no significant differences were observed in any of
    the clinical biochemical parameters between treated and untreated
    animals of either sex.  No histopathological examination was
    performed (Majnarich 1988).

         In a 28-day toxicity study spray dried  Dunaliella bardawil
    powder was orally administered to rats (5/group/sex) at dosages of
    0.5 and 2.5 g  Dunaliella bardawil/kg bw/dy.  The control animals
    (only) received 0.5 % aqueous solution of sodium CMC, which was used
    as the vehicle for the preparation of the test article suspension. 
    Toxicological parameters recorded included food consumption, body
    weight gain, urinalysis, ophthalmoscopy, haematology, serum
    biochemistry, organ weights and histopathological examination.

         In the males of the 2.5 g  Dunaliella bardawil/kg bw/dy  group
    a significant increase in the relative weight of the kidneys was
    noticed.  Except slight changes in the thymus and the kidneys in a
    few animals of the 2.5 g  Dunaliella bardawil/kg bw/dy group, no
    histopathological changes were reported.  Although the effects were
    observed mainly in the kidneys, it was suggested by the authors that
    the dosage of 2.5 g  Dunaliella bardawil/kg bw/dy was a NOEL
    (Furahashi, 1989).

    2.2.3  Long-term toxicity/carcinogenicity studies

         No information available.

    2.2.4  Reproduction studies

         The safety of the alga  Dunaliella bardawil for food use was
    evaluated in a multigeneration study with rats.  Four generations
    were raised on diets containing 0, 50 and 100 g/kg of dehydrated
     D. bardawil.  The caloric value of the diets with the  Dunaliella
     bardawil preparations was adapted by lowering the amount of

    starch.  Each experimental group comprised 10 males and 20 females. 
    Starting with an F0-generation 3 other generations (F1 - F3)
    were raised.  The rats of the F0-generation were kept on the
    different diets for 1 year and 5 male and 5 female rats were studied
    for general toxicological effects.  No significant differences were
    observed between the rats consuming algae and the controls, of any
    generation, in general appearance, behaviour, growth, reproductive
    performance or gross pathology.  The only effect of  D. bardawil
    powder observed was a significantly increased relative kidney
    weights.  The blood chemistry and haematology of the
    first-generation animals, after 1 year on the diets, showed no
    appreciable differences between the experimental and control
    animals.  The only differences in histopathology observed were a
    decrease in some chronic inflammations, a slightly higher frequency
    of metaplasia of the renal pelvis epithelium with ectopic
    nephrocalcinosis in the renal papillae and an increased frequency of
    focal bronchopneumonia in rats fed 10 g algae/kg feed when compared
    with the controls.  The latter effect may be attributed to the
    powdery nature of the algal diet.  Although at dose levels effect
    were recorded on the kidneys and which were not explained, the
    authors concluded that this multigeneration feeding study may be
    indicative of the safety of  D. bardawil for human consumption
    (Mokady  et al., 1989).

    2.2.5  Special studies on genotoxicity

         A mutagenicity study (Ames test) in  Salmonella typhimurium TA
    98, TA 100, TA 1535 and TA 1537 and  Escherichia coli WP2 uvrA both
    with and without activation by a liver microsomal S-9 mix was
    performed at dose levels of 312.5, 625, 1 250, 2 500 and 5 000 g
     Dunaliella bardawil paste/plate.   Dunaliella bardawil was not
    mutagenic in any strain (Aruga, 1988).

    2.3  Observations in humans

         Nine subjects were maintained on a low-carotene diet for two
    weeks and serum carotene levels were then determined.  For the next
    ten days the volunteers took a daily dose of the powdered algal
    preparation providing 75 000 IU -carotene (approx 135 mg) in
    capsule form.  Serum carotene was measured on days 7 and 10 of
    treatment.  There was considerable interindividual variation in
    response to the same dose of carotenes, both in absolute values and
    in the treatment-dependent increase in serum concentration of
    carotene.  In six of the subjects the serum level of carotene
    continued to rise between the seventh and tenth day of the study
    while in three others there was a slight fall in this period.  One
    subject with normal serum carotene levels at the outset showed
    virtually no response to treatment.  No adverse effects due to
    ingestion of the algal preparation were reported (Cyanotech, 1988).

    ALGAL CAROTENE PREPARATIONS
    VEGETABLE OIL EXTRACT OF  DUNALIELLA SALINA
    (syn  DUNALIELLA BARDAWIL and DUNALIELLA KONA)

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion.

         In an experiment fully described in section 2.1.1 describing
    results with spray dried concentrated, lyophilized or dehydrated
    preparations of  Dunaliella, male weanling CD rats were fed a
    retinol-deficient diet  ad libitum.  After depletion, rats were
    allocated to groups and fed a retinol-deficient diet supplemented
    with one of: retinol; all- trans -carotene; lyophilized
     Dunaliella; maize oil extract of  Dunaliella; and no
    supplementation.  After seven days repletion, livers were taken for
    analysis of retinol, retinol isomers and -carotene.  The liver
    analysis revealed a comparable content of retinol related to dose of
    carotene, irrespective of source i.e. synthetic, algal biomass or
    algal oil extract.  It was concluded that dried  Dunaliella bardawil
    or an oil extract of the alga can serve as a dietary natural
    -carotene source which can satisfy the total requirement of retinol
    in rats (Ben-Amotz  et al. 1988).

    2.1.2  Biotransformation

         No information available.

    2.1.3  Effects on enzymes and other biochemical parameters

         No information available.

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         No information available.

    2.2.2  Short-term toxicity studies

         No information available.

    2.2.3  Long-term toxicity/carcinogenicity studies

         No information available.

    2.2.4  Reproduction studies

         No information available.

    2.2.5  Special studies on genotoxicity

         The commercial, carotene-rich corn oil extract of  Dunaliella
     salina was inactive in an  in vitro primary hepatocyte
    unscheduled DNA synthesis assay (Cifone 1987).

         The extract was negative in an assay of forward mutation at the
    HGPRT locus in cultured Chinese hamster ovary cells, with or without
    metabolic activation with rat liver S9 fraction.  A dose-related
    cytotoxicity was noted at concentrations above 2.0 l/ml without S9
    and above 10.0 l/ml in the presence of S9 (Young 1987).

         The material was not mutagenic in the  Salmonella/microsome
    assay (Ames test) with  Salmonella typhimurium strains TA-1535,
    TA-1537, TA-1538, TA-98 and TA-100 with or without metabolic
    activation (Jagannath 1987).

         In an  in vivo mouse micronucleus assay using adult ICR mice,
    the commercial carotene extract did not induce a significant
    increase in micronuclei in bone marrow polychromatic erythrocytes
    (Ivett 1987).

    2.3  Observations in humans

         After a depletion period of 10 days on a low-carotene diet, 12
    male and 20 female healthy adults were randomly assigned to one of
    five treatment groups.  Two groups received capsules of carotene
    obtained by vegetable oil extraction of  Dunaliella salina
    providing -carotene at levels of 8 or 24 mg and alpha-carotene at
    levels of 1.1 or 3.2 mg respectively.  Two further groups received
    carrots (69.1 or 207.3 g respectively) that provided a similar
    amount of -carotene to the  Dunaliella salina extract groups; the
    corresponding amounts of alpha-carotene were 6.3 and 18.9 mg
    respectively.  A fifth group received placebo capsules.  The
    subjects received the treatment for seven days and then underwent
    another depletion phase of 7 days.

         Treatment with carotene capsules or carrots led to an expected
    increase in serum alpha- and -carotenes, with the higher dose
    treatments being less efficient per mg carotene consumed.  The
    encapsulated algal carotenes were more efficient at raising serum
    values per mg fed, consistent with other reports that carotenes are
    better absorbed from oily solution than a vegetable matrix (Jensen
     et al. 1985).

         In a study on the bioavailability of cis- and trans--
    carotenes, 16 healthy adults, who had been on a low-carotene diet
    for ten days, were fed either -carotene extracted from  Dunaliella
     salina alga, containing approximately equal amounts of all-trans-
    -carotene and 9-mono-cis--carotene, or -carotene in the form of
    fresh carrots containing predominantly trans--carotene, or avocado

    oil-placebo capsules.  Subjects were randomly divided into three
    groups: they consumed daily in a single dose either 3 -carotene
    capsules (24 mg -carotene), 207.3 g carrots (24 mg -carotene); or
    3 -carotene free placebo capsules for seven days.  HPLC
    determinations of serum trans-cis -carotene ratios showed trans
    -carotene to be the predominate serum isomer before and during all
    treatments.  Serum trans--carotene concentrations were
    significantly increased in the -carotene capsules and carrot
    groups.  Cis--carotene concentrations were increased in the carrot
    and placebo groups.  However, the serum isomer increments for those
    taking -carotene capsules and carrots strongly favoured trans-
    -carotene over cis--carotene.  These data demonstrate a
    predominant absorption of intact trans--carotene over intact cis-
    -carotene into human serum even when approximately equivalent
    amounts of these isomers were ingested.  This selective absorption
    of intact -carotene isomers might be a factor in their biopotency
    in humans (Jensen  et al., 1987).

    CAROTENE EXTRACTS FROM VEGETABLES
    (Carrots, alfalfa and vegetable oil)

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

         No information available.

    2.2  Toxicological studies

         No information available.

    2.3  Observations in humans

         The acute effects of consuming alpha- and -carotene from
    carrots on serum alpha-carotene and -carotene levels were
    investigated in 17 adult subjects (18-58 years of age).  After a
    10-day low-carotene diet, the subjects were randomized into three
    groups based on day 6 -carotene levels.  On day 11, fasting
    baseline blood was drawn.  Either 3 carrots, 1 carrot or 3 placebo
    capsules were then consumed following a low-carotene breakfast. 
    Blood was drawn 1, 2, 3, 4, 5, 7 and 24 hours post-treatment and
    alpha- and -carotene levels were determined by HPLC.  Treatment of
    3 carrots yielded significantly greater peak alpha- and -carotene
    levels in serum at 5 hours post-treatment than did treatments with 1
    carrot or 3 placebos.  These results suggest the best condition for
    drawing blood samples to assess the serum carotene status of adults
    is at fasting state and that significant alterations in serum can
    occur within 5 hours of a carotene rich meal (Jensen  et al.,
    1986).

    3.  COMMENTS

         Few new toxicological data have become available since the
    previous review by the Committee (Annex 1, reference 88).  There
    were no data from long-term toxicity or teratogenicity studies,
    although a multigeneration study on dehydrated  Dunaliella bardawil
     (= salina) in rats did not reveal any adverse effects on
    reproductive performance or gross fetal morphology.  However, a NOEL
    was not identified in this study, as animals of the F0-generation
    maintained on diets containing 5% and 10% algal carotene for one
    year showed renal pathological changes.  In addition, there was
    focal bronchopneumonia at the higher level.  Although the renal
    changes (metaplastic changes in the pelvic epithelium and
    nephrocalcinosis) might have been due to nutritional imbalance, this
    was not clearly established.  The focal bronchopneumonia observed in
    the lung at the higher dose level may have been associated with
    inhalation of powdered diets, although both low-dose and control
    rats also received powdered diets without showing similar effects. 
    The Committee considered that the available short-term toxicity
    studies inadequate for establishing an ADI because of the small
    numbers of animals tested, lack of or inadequate histopathological
    examination, or inadequate reporting.

         There were virtually no systematic toxicological studies
    available on the oil-extract of alga.  Available data on the dried
    material could not be extrapolated to the oil extract since the
    specifications are quite different and lipophilic materials may have
    been concentrated during the oil extraction process.

    4.  EVALUATION

         The Committee considered the data inadequate to establish an
    ADI for the dehydrated algal carotene preparations or for the
    vegetable oil extracts of  Dunaliella salina.  There is no history
    of use of  Dunaliella algae as food.

         No relevant toxicological data on vegetable extracts were
    available.  However, the Committee concluded that there was no
    objection to the use of vegetable extracts as colouring agents,
    provided that the level of use did not exceed the level normally
    present in vegetables.  Implicit in this conclusion is that the
    extracts should not be made toxic by virtue of the concentration of
    toxic compounds (including toxicants naturally occurring in the
    vegetables) nor by the generation of reaction products or residues
    of a nature or in such amounts as to be toxicologically significant

    5.  REFERENCES

    ARUGA, F. (1987).  Acute oral toxicity study on  Dunaliella bardawil
    spray dried powder in mice.  Nihon Bioresearch  Center Inc. 
    Hashima, Gifu, Japan, as submitted to WHO by Nikken Sohonsha
    Corporation, Hashim-City, Japan.

    ARUGA, F. (1988).  Mutagenicity test of  Dunaliella bardawil paste
    with  Salmonella typhimurium and  Escherichia coli.  Nihon
    Bioresearch Center Inc.  Hashima, Gifu, Japan, as submitted to WHO
    by Nikken Sohonsha Corporation, Hashim-City, Japan.

    BEN-AMOTZ, A., EDELSTEIN, S. & AVRON, M. (1986) Use of the
    -carotene rich alga  Dunaliella bardawil as a source of retinol. 
     Brit. Poultry Sci., 27, 613-619

    BEN-AMOTZ, A., MOKADY, S. & AVRON, M. (1988) The -carotene-rich
    alga  Dunaliella bardawil as a source of retinol in a rat diet. 
     Brit. J. Nutr., 59, 442-449.

    CIFONE, M.A. (1987) Mutagenicity test on EK 87-0048 B-CAT in the rat
    primary hepatocyte unscheduled DNA synthesis assay.  Unpublished
    report of Hazleton Laboratories America Inc. Submitted to WHO by
    Eastman Kodak Co., Rochester, NY, USA.

    CYANOTECH (1988) Ten-day Konatene (TM) feeding study: effects on
    serum beta-carotene levels.  Unpublished summary report submitted to
    WHO by Cyanotech Corporation, Woodinville, Washington, USA.

    FURUHASHI, T (1989) Twenty-eight-day oral subacute toxicity study on
     Dunaliella bardawil.  Nihon Bioresearch Center Inc., Hashima,
    Gifu, Japan, as submitted to WHO by Nikken Sohonsha Corporation,
    Hashima-City, Japan.

    GHAZI, A., DE LUMEN, B. & OSWALD, W.J. (1992).  Comparative
    bioavailability of beta-carotene from  Dunaliella salina, Dunaliella
     salina extract, carrot extract and synthetic beta-carotene. 
    Report submitted to WHO by Microbio Resources, Inc., San Diego, CA
    USA.

    IVETT, J.L. (1987) Mutagenicity test on EK 87-0047, corn oil control
    and EK 87-0048, B-CAT in the  in vivo mouse micronucleus assay. 
    Unpublished report of Hazleton Laboratories America Inc. submitted
    to WHO by Eastman Kodak Co., Rochester, NY, USA.

    JAGANNATH, D.R. (1987) Mutagenicity test on EK 87-0048 B-CAT in the
    Ames Salmonella/microsome reverse mutation assay.  Unpublished
    report of Hazleton Laboratories America Inc. submitted to WHO by
    Eastman Kodak Co., Rochester, NY, USA.

    JENSEN, C.D., PATTISON, T.S., SPILLER, G.A., WHITTAM, J.H. & SCALA,
    J. (1985) Repletion and depletion of serum alpha and beta carotene
    in humans with carrots and an algae-derived supplement.   Acta
     Vitaminol. Enzymol., 7, 189-198.

    JENSEN, C.D., SPILLER, G.A., PATTISON, T.S., WHITTAM, J.H. & SCALA,
    J. (1986).  Acute effects of dietary carotenes on serum alpha and
    beta carotene in humans.  Nutr. Rep.  Int., 33, 117-122.

    JENSEN, C.D., HOWES, T.W., SPILLER, G.A., PATTISON, T.S., WHITTAM,
    J.H. & SCALA, J. (1987).  Observations on the effects of ingesting
     cis- and  trans-beta-carotene isomers on human serum
    concentrations.   Nutr. Rep. Int., 35, 413-422.

    LOCK, S. (1985) Fourteen days oral rat testing using dried
     Dunaliella cells, BioMed No.  4476.  Unpublished summary report of
    Biomed Research Laboratories Inc. submitted to WHO by Cyanotech
    Corporation, Woodinville, Washington, USA.

    MAJNARICH, J.J. (1988) Subchronic oral toxicity (12 week) study of
    algal beta carotene fed to male and female Sprague-Dawley rats. 
    Unpublished report of Biomed Research Laboratories Inc. submitted to
    WHO by Cyanotech Corporation, Woodinville, Washington, USA.

    MOKADY, S., ABRAMOVICI, A. & COGAN, U. (1989) The safety evaluation
    of  Dunaliella bardawil as a potential food supplement.   Fd. Chem.
     Toxic., 27, 221-226.

    NAGASAWA, H., FUJII, Y., YAMAMOTO, K., KONOSHI, R. & BEN-AMOTZ, A.
    (1989).  No deleterious side-effects on mammary growth and endocrine
    parameters of chronic ingestion of beta-carotene-rich alga
     Dunaliella bardawil in virgin mice in comparison with synthetic
    all- trans beta-carotene.   The Cancer Journal, 2, 391-394.

    YOUNG, R.R. (1987) Mutagenicity test on EK 87-0048 B-CAT in the
    CHO/HGPRT forward mutation assay.  Unpublished report of Hazleton
    Laboratories America Inc. submitted to WHO by Eastman Kodak Co.,
    Rochester, NY, USA.


    See Also:
       Toxicological Abbreviations