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    GELLAN GUM

    First draft prepared by Dr F.S.D. Lin,
    Division of Toxicological Review and Evaluation,
    Center for Food Safety and Applied Nutrition,
    US Food and Drug Administration.

    1.  EXPLANATION

         Gellan gum has not been previously evaluated by the Joint
    FAO/WHO Expert Committee on Food Additives.

         Gellan gum is a high molecular weight polysaccharide gum
    produced as a fermentation product by a pure culture of  Pseudomonas
     elodea.  The production organism is an aerobic, gram-negative
    bacterium, which has been very well characterized and demonstrated
    to be non-pathogenic.  Chemical structure of the polysaccharide has
    been determined.  It has a tetrasaccharide repeat unit consisting of
    two glucose (Glc) residues, one glucuronic acid (GlcA) residue, and
    one rhamnose (Rha) residue:

    -> 3)-D-G1c-(1->4)--D-G1cA-(1->4)--D-G1c-(1->4)-L-Rha-(1->

         The glucuronic acid is neutralized by the presence of
    potassium, calcium, and magnesium ions.  The relative concentrations
    of these ions will control the physical properties of the gum
    material such as gel strength, melting point and setting point.  The
    molecular weight of the polysaccaride is greater than 70 000 with
    95% above 500 000.  The gum has been proposed for use as a
    stabilizer and thickener in foods.

         There are three basic forms of gellan gum product which have
    been characterized and are distinguished by their 1) polysaccharide
    content, 2) the percent of o-acetyl substitution on the
    polysaccharide and 3) the protein content (including nucleic
    residues and other organic nitrogen sources).  

         It is noted that a relatively pure (>95% polysaccharide) non-
    acetylated gum product was used in the acute toxicity studies, the
    13-week oral rat study and the genotoxicity studies.  For the
    remaining toxicological studies, a blend of 5 products with lower
    purities and varied degrees of acetylation was used.  This blend,
    which contained 58.5% polysaccharide, was intended to represent the
    complete range of possible compositions of the gum product and was
    considered as the "worst case" in terms of purity.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion

         The absorption, distribution and excretion of gellan gum was
    studied using a dually radiolabelled (3H and 14C) preparation. 
    The use of dual labelling allowed simultaneous quantitation of both
    polysaccharide and "protein" fractions of gellan gum.

         The gellan gum was prepared in separate fermentations using
    3H-glucose and 14C-glucose as carbon source.  The 3H product was
    subjected to multi-stage purification process to give a relatively
    pure 3H-polysaccharide.  This was added to the media of the 14C
    fermentation, which was then precipitated in isopropanol to yield a
    product with the polysaccharide fraction labelled with both isotopes
    and the non-polysaccharide (or "protein") fraction labelled only
    with 14CO2.

         One male and one female Sprague-Dawley rat were gavaged with
    single doses of the 3H/14C-gellan gum (ca. 960 mg/kg; ca. 4 Ci). 
    Expired air was collected for 24 hours after dosing.  Less than
    0.55% of the given radioactivity was detected as 14C.

         Four male and 3 female Sprague-Dawley rats were dosed with
    single gavage dose of 3H/14C-gellan gum (ca. 870 mg/kg; 2.9 - 4.1
    Ci 14C; 0.7 - 0.9 Ci 3H).  Urine and faeces were collected for 7
    days, at which time the animals were sacrificed and their tissues
    analyzed for residual radioactivity.  Females excreted 86.8% and
    1.9% of the given 14C in their faeces and urine, respectively. 
    Males excreted 86% of the dosed 14C in the faeces and 3.3% in the
    urine.  Females excreted 4.1% of the dosed 3H in their urine and
    100.1% in their faeces, while males excreted 3.6% of the total 3H
    in their urine and 99.6% in their faeces.  In all animals, the
    activities of 3H in tissues (blood, brain, liver, kidney, lung,
    muscle, skin, heart and carcass) were too low to be quantitated
    accurately.  Tissue and carcass radioactivity for 14C averaged 3.8%
    of dose for male rats and 3.0% of dose for female rats.

         A male and four female Sprague-Dawley rats were gavaged with
    about 1 g/kg of radiolabelled gellan gum and blood samples collected
    from the tail vein at different time intervals over a 7-day period. 
    Data were reported as 14C dmp/ml blood (3H dmp/ml blood was not
    reported).  The peak level of radioactivity, which amounted to about
    0.4% of the administered radioactivity, occurred about 5 hours after
    dosing (Selim, 1984a).

    2.2  Toxicological studies

    2.2.1  Acute toxicity
                                                                      
                                       LD50
    Species   Sex    Route          (mg/kg b.w.)      Reference
                                                                      

    Rat       M&F    oral           >5000             Wolfe & Bristol,
                                                      1980

              M&F    inhalation     >5.09 mg/l        Coate et al., 
                                                      1980

                                                                      

         Gellam gum is practically non-toxic to rats when administered
    as a single large dose (5 g/kg b.w.) in diet or via gavage.

    2.2.2  Short-term studies

    2.2.2.1  Rat

         Male and female Sprague-Dawley rats (20/sex/group) were fed
    dietary levels of GG ranging from 0-6% for 13 weeks.  Although the
    animals on this study experienced symptoms of a sialodacryoadenitis
    viral infection, all animals survived treatment and there were no
    adverse effects associated with the feeding of GG (Batham  et al.,
    1983).

    2.2.2.2  Monkey

         Prepubertal rhesus monkeys (2/sex/group) were dosed by oral
    gavage with GG at levels of 0, 1, 2 or 3 g/kg/day for 28 days. 
    There were no overt signs of toxicity reported (Selim, 1984b).

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Mouse

         Groups of 50 male and 50 female Swiss Crl mice were fed GG
    admixed in the diet at 0, 1,0, 2.0 and 3.0% for 96 and 98 weeks for
    males and females, respectively.  All animals were examined twice
    daily for mortality and morbidity.  Physical examination for the
    presence of palpable masses was initiated on a weekly basis starting
    in week 26.  Bodyweights and food consumption were measured for 7-
    day periods on a weekly basis for the first 26 weeks of treatment
    and every 2 weeks thereafter.  At necropsy, a complete gross
    pathological examination was performed on the following organs and
    tissues of the animals from the control and 3.0% groups: adrenals,

    aorta (thoracic), bone (sternum), brain (fore-, mid- and hind-),
    caecum, colon, duodenum, epididymis, oesophagus, eyes, Harderian
    gland, heart, ileum, jejunum, kidneys, lacrimal gland, liver (sample
    of 2 lobes), lung (sample of 2 lobes), lymph nodes (mandibular and
    mesenteric), mammary gland (inguinal), nasal turbinates, optic
    nerves, ovaries, pancreas, pituitary, prostate, rectum, salivary
    gland, sciatic nerve, seminal vesicles, skeletal muscle, skin,
    spinal cord, spleen, stomach, testes, thymus, thyroid lobes (and
    parathyroids,), tongue, trachea, urinary bladder, uterus, vagina,
    Zymbal's gland and all gross lesions.  Only the liver, kidneys,
    ovaries, testes, adrenals, pituitary, lungs and heart were examined
    for animals of the 1.0 and 2.0% groups.  There were no effects
    attributable to the feeding of GG on either body weight gain or food
    consumption.  There were no neoplastic or non-neoplastic changes
    which were associated with the feeding of GG (Batham  et al., 
    1987).

    2.2.3.2  Rat

         Groups of 50 F1 generation Sprague-Dawley rats of each sex
    were exposed to GG  in utero and continued on GG diets for
    approximately 104 weeks.  The dietary levels of GG were 0, 2.5, 3.8
    and 5.0%.  The rats were observed daily for the first 4 weeks of
    treatment and weekly thereafter for clinical signs of toxicity. 
    Individual bodyweights and food consumption were measured on a
    weekly basis for the first 26 weeks of treatment and every two weeks
    thereafter.  Funduscopic and biomicroscopic examinations were
    conducted on the control and 5% groups during weeks 1, 13, 26, 52,
    78 and 103.  Clinical chemistry and haematological samples were
    collected at weeks 13, 25, 39 and 51.  After 104 weeks,
    ophthalmoscopic examinations, haematology, clinical chemistries and
    organ weight data revealed no changes which could be attributed to
    the feeding of GG.  Survival of male treated rats was poor when
    compared to controls whereas female treated rats exhibited better
    survival than their concurrent controls.  Male rats, fed GG at the
    3.8 and 5.0% dietary levels, exhibited lower bodyweights after 76
    weeks.  The initial bodyweights were 5.2 and 3.4% lower than the
    control values for the 3.8% and 5.0% dietary levels, respectively. 
    The authors concluded that in spite of the initial bodyweight
    deficit, the growth pattern for these treated groups was identical
    to that of the control.  In addition, this effect was not seen in
    either the females or any other species tested.  There is no basis
    to suggest that the lower bodyweights, observed in the male rats,
    are indicative of toxicity.

         Organs and tissues as those listed in the above mouse study
    were examined for histopathological changes at study termination. 
    There were no neoplastic or non-neoplastic changes that could be
    associated with the feeding of GG.  The authors concluded that under

    the conditions of this bioassay, GG was non-carcinogenic to Sprague-
    Dawley rats (Batham  et al., 1985).

    2.2.3.3  Dog

         Diets containing 0, 3, 4.5 and 6% GG were fed to groups of 5
    beagle dogs per sex for a period of 52 weeks.  The dogs were
    observed daily for clinical signs of toxicity and were measured for
    bodyweights and food consumption.  Ophthalmoscopic examinations were
    performed during pretreatment and after 12 , 24, 39 and 51 weeks. 
    Haematology and clinical chemistry were measured during pretreatment
    and after 6, 13, 25, 39 and 50 weeks.  After 52 weeks all animals
    were killed and grossly examined.  The following organs and tissues
    were removed, processed and examined for histopathological lesions:
    adrenals, aorta, bone and marrow, brain, caecum, colon, duodenum,
    epididymis, oesophagus, eyes, gall bladder, heart, ileum, jejunum,
    kidneys, liver, lungs, lymph nodes, mammary gland, optic nerves,
    ovaries and ovariducts, pancreas, pituitary, prostate, rectum,
    salivary gland, sciatic nerve, skeletal muscle, skin, spinal cord,
    spleen, stomach, testes, thymus, thyroid and parathyroid, tongue,
    trachea, urinary bladder and uterus.

         All animals survived treatment.  Food intake was higher in the
    treated groups compared to the controls.  There were no adverse
    effects associated with the feeding of GG to beagle dogs for a
    period of one year (Batham  et al., 1986).

    2.2.4  Reproduction studies

         Groups of 26 male and 26 female CD (Sprague-Dawley) rats were
    administered GG in their diets at doses of 0, 2.5, 3.8 or 5.0%. 
    Males were treated for 70 days prior to mating and for three weeks
    after mating.  Females were treated for 14 days prior to mating and
    throughout mating, gestation and lactation.  Selection was made for
    the pups (F1) of this mating and they were allowed to mature and
    were mated to form  the F2 generation.

         There was no treatment-related effect on mating or fertility
    index, conception rate, length of gestation, length of parturition,
    number of live pups, number of dead pups, post-implantation loss
    index, survival index on day 4, 7, 14 or 21 or lactation index for
    any of the generations (Robinson  et al., 1985a).

    2.2.5  Teratology studies

         GG was fed to groups of 25 pregnant female Sprague-Dawley rats
    at dietary levels of 0, 2.5, 3,8 or 5.0% during days 6-15 of
    gestation.  GG had no fetotoxic or teratogenic effects on rats when
    ingested in the diet at levels up to 5.0% ( Robinson et al., 
    1985b).


        2.2.6  Genotoxicity studies

    Results of genotoxicity assays on gellan gum
                                                                                                    

    Test system         Test object         Concentration of         Results       Reference
                                            gellan gum                             
                                                                                                    

    Ames test (1)       S. typhimurium      10, 30, 100, 300 and     Negative      Robertson et al., 
                        TA98, TA100         1000 g/plate                          1985a
                        TA1535 TA1537                                              
                        TA1538                                                     

    DNA repair test     Rat hepatocyte      3, 5, 10 & 20 mg/ml      Negative      Robertson et al., 
                                                                                   1985a,b

    V-79/HGPRT          Chinese hamster     3, 5, 10 & 20 mg/ml      Negative      Robertson et al., 
                        lung fibroblasts                                           1985c


                                                                                                    

    (1) Both with and without rat liver S-9 fraction.
    

    2.3  Observations in humans

         Five female volunteers and five male volunteers, all normal in
    health and free from gastrointestinal disease, participated in the
    clinical study.  Following a 7-day control period, each of the
    volunteers consumed the test substance at a daily dose level of 175
    mg/kg for 7 days, then the dose was increased to 200 mg/kg/day for a
    further 16 days.  Plasma biochemistry parameters, haematological
    indices, urinalysis parameters, blood glucose and plasma insulin
    concentrations and breath hydrogen concentrations were monitored on
    the first day of the control period and repeated on the last day of
    the treatment period.

         The authors concluded that the ingestion of gellan gum at the
    given dose levels caused no adverse dietary nor physiological
    effects in any of the volunteers on the study.  There were no
    allergenic nor other subjective untoward manifestations, reported by
    or observed in any of the human subjects.  The ingestion of gellan
    gum, at the stated daily intake levels, did not cause any adverse
    toxicological effects.  However, gellan gum does act as a faecal
    bulking agent, increases faecal bile acid, decreases faecal neutral
    sterols, and decreases serum cholesterol (Eastwood  et al., 1987).

    3.  COMMENTS

         Gellan gum was shown to be poorly absorbed and did not cause
    any deaths in rats which received a single large dose (5 g per kg of
    body weight) in the diet or by gavage.  Short-term (90-day) exposure
    of rats to gellan gum at levels up to 60 g/kg in the diet did not
    cause any adverse effects.  In a 28-day study in prepubertal
    monkeys, no overt signs of toxicity were observed at the highest
    dose level of 3 g per kg of body weight per day.  In reproduction
    and teratogenicity studies in rats in which gellan gum was given at
    dose levels up to 50 g/kg in the diet, there was no evidence of
    interference with the reproductive process, and no embryotoxic or
    developmental effects were observed.  Gellan gum was also shown to
    be non-genotoxic in a battery of standard short-term tests.

         In a study in dogs, which were treated for 1 year at dose
    levels up to 60 g/kg in the diet, there were no adverse effects that
    could be attributed to chronic exposure to gellan gum.  In long-term
    carcinogenicity studies, gellan gum did not induce any adverse
    effects in mice or rats at the highest dose levels of 30 g/kg and 50
    g/kg in the diet, respectively.

         Results from a limited study on tolerance to gellan gum in
    humans indicated that oral doses of up to 200 mg per kg of body
    weight administered over a 23-day period did not elicit any adverse
    reactions, although faecal bulking effects were observed in most
    subjects.

    4.  EVALUATION

         The Committee allocated an ADI "not specified" to gellan gum,
    and pointed out that its potential laxative effect at high intakes
    should be taken into account when it is used as a food additive
    (Annex I, ref. 88, Section 2.2.3).

    5.  REFERENCES

    BATHAM, P., RAINEY, S., BIER, C., LOSOS, G., OSBORNE, B.E. &
    PROCTER, B. (1983).  A 13-week toxicity study of a polysaccharide
    gum (K9A50) during dietary administration to the albino rat. 
    Unpublished project No. 81274 from Bio-Research Laboratories Ltd.,
    Montreal, Canada.  Submitted to WHO by Kelco (Division of Merck &
    Co., Inc.), San Diego, CA, USA.

    BATHAM, P., PINSONNEAULT, R.T. & PROCTER, B.G. (1985).  An in
    utero/chronic toxicity/carcinogenicity study of gellan gum
    administered in the diet to the rat (in utero phase).  Unpublished
    project No. 81835 from Bio-Research Laboratories Ltd., Montreal,
    Canada.  Submitted to WHO by Kelco (Division of Merck & Co., Inc.),
    San Diego, CA, USA.

    BATHAM, P., KALICHMAN, S.G. & OSBORNE, B.E. (1986). A 52-week oral
    toxicity study of gellan gum in the beagle dog. Unpublished project
    No. 81779 from Bio-Research Laboratories Ltd., Montreal, Canada.
    Submitted to WHO by Kelco (Division of Merck & Co., Inc.), San
    Diego, CA, USA.

    BATHAM, P., ENGEL, D., OSBORNE, B.E. (1987).  A dietary
    carcinogenicity study of gellan gum in the albino mouse. 
    Unpublished project NO. 81833 from Bio-Research Laboratories Ltd.,
    Montreal, Canada.  Submitted to WHO by Kelco (Division of Merck &
    Co., Inc.), San Diego, CA, USA.

    COATE, W.B., KEENAN, D.L., VOELKER, R. & HARDY, R.J. (1980).  Acute
    inhalation toxicity study in rats.  Unpublished project No. 2123-105
    from Hazelton Laboratories America, Inc., Vienna, VA, USA. 
    Submitted to WHO by Kelco (Division of Merck & Co., Inc.), San
    Diego, CA, USA.

    EASTWOOD, M.A., BRYDON, W.G., & ANDERSON, D.M.W. (1987).  The
    dietary effects of gellan gum in humans.  Unpublished report from
    Wolfson Gastrointestinal Laboratory, Edinburgh, U.K.  Submitted to
    WHO by Kelco (Division of Merck and Co., Inc.), San Diego, CA, USA.

    ROBERTSON, R.T., NICHOLS, W.W., BOKELMAN, D.L. & BRADLEY, M.O.
    (1985a).  Microbial mutagenicity test.  Unpublished report No. 83-
    870 from Merck Sharp & Dohme Research Laboratories, West Point, PA,
    USA. Submitted to WHO by Kelco (Division of Merck & Co., Inc. ), San
    Diego, CA, USA.

    ROBERTSON, R.T., NICHOLS, W.W. & BOKELMAN, D.L. (1985b). 
    Unscheduled DNA synthesis in rat hepatocytes: Auto-radiographic
    assay.  Unpublished report No. 84-8403 from Merck Sharp & Dohme
    Research Laboratories, West Point, PA, USA.  Submitted to WHO by
    Kelco (Division of Merck & Co., Inc.), San Diego, CA, USA.

    ROBERTSON, R.T., NICHOLS, W.W. & BOKELMAN, D.L. (1985c).  Gellan
    Gum: V-79 mammalian cell mutagenesis.  Unpublished report Nos. 84-
    8531 and 83-8530 from Merck Sharp & Dohme Research Laboratories,
    West Point, PA, USA.  Submitted to WHO by Kelco (Division of Merck &
    Co., Inc.), San Diego, CA, USA.

    ROBINSON, K., THIBAULT, C. & PROCTER, B.G. (1985a).  A two
    generation reproduction study of gellan gum administered in the diet
    to the rat.  Unpublished project No. 81834 from Bio-Research
    Laboratories LTD, Montreal, Canada.  Submitted to WHO by Kelco
    (Division of Merck & Co., Inc.), San Diego, CA, USA.

    ROBINSON, K., THIBAULT, C. & PROCTER, B.G. (1985b).  A teratology
    study of gellan gum administered in the diet to the rat. 
    Unpublished project No. 81890 from Bio-Research Laboratories LTD,
    Montreal, Canada.  Submitted to WHO by Kelco (Division of Merck &
    Co., Inc.), San Diego, CA, USA.

    SELIM, S. (1984a).  Rat balance study, tissue distribution and blood
    level of 14C and 3H labelled gellan gum.  Unpublished study No.
    KE-162r from Primate Research Institute, Holloman Air Force Base,
    New Mexico, USA.  Submitted to WHO by Kelco (Division of Merck &
    Co., Inc.), San Diego, CA, USA.

    SELIM, S., FULLER, G.B. & BURNETT, B. (1984b). A 28-day subchronic
    toxicity study in rhesus monkeys. Unpublished project No. KE-170m
    from Primate Research Institute. Holloman Air Force Base, New
    Mexico, USA. Submitted to WHO by Kelco (Division of Merck & Co.,
    Inc.), San Diego, CA, USA.

    WOLFE, G.W., & BRISTOL, B.A. (1980).  Acute oral toxicity study in
    rats.  Unpublished project NO. 2123-103 from Hazelton Laboratories
    America, Inc. Vienna, VA, USA.  Submitted to WHO by Kelco (Division
    of Merck & Co., Inc.), San Diego, CA, USA.


    See Also:
       Toxicological Abbreviations
       GELLAN GUM (JECFA Evaluation)