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    CANDELILLA WAX

    First draft prepared by
    Dr D.L. Grant
    Bureau of Chemical Safety
    Health and Welfare
    Ottawa, Ontario, Canada

    1.  EXPLANATION

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

         Candelilla wax is obtained from candelilla plants  (Euphorbia
     antisiphilitica, Euphorbia cerifers, Pedilanthus pavonis) which
    are found in the dry regions of northern Mexico and to a lesser
    extent in southern Texas, Arizona and California in the United
    States. Candelilla wax is obtained through extraction from the
    plants by immersion in a tank containing boiling water acidified
    with sulfuric acid. Candelilla wax is a hard and brittle wax. It is
    composed of about 20-29% wax esters, 12-14% alcohols and sterols,
    49-50% hydrocarbons, 7-9% free acids, 2-3% moisture and 1% mineral
    matter. The chemical and physical properties of the wax vary with
    the age of the plant and the year in which it is collected. The wax
    is insoluble in water but soluble in acetone, chloroform, benzene,
    and other organic solvents (Rogers, 1978; FDA, 1982; ACT, 1984;
    Ashraf-Khorassani & Taylor, 1990).

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

         No information available

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

                                                                                      

    Species         Sex           Route     LD50            Reference
                                           (mg/kg bw)
                                                                                  

    Rat             Not defined   oral     > 5000           ACT, 1984
                                                                                  
    
    2.2.2  Short-term studies

    2.2.2.1   Rats

         Groups of 12 male and 12 female weanling Wistar rats, weighing
    40-50 g, were fed a diet containing a mixture "(Product 12)" of gum
    base and candelilla wax (composition not defined), at concentrations
    of 0, 3 or 5%, equivalent of 0, 590 or 980 mg/kg bw/day for 8 weeks.
    No significant differences were reported between groups, regarding
    survival, body weight gains, food and water intake, urinalysis
    (parameters measured not identified) haematology (parameters
    measured not identified) nor gross pathology (Harrisson, 1946).

         Groups of weanling Wistar rats (12 rats/sex/group) were fed
    diets containing a 1:1 mixture of candelilla wax "(Product 16)" and
    butadiene-styrene polymer "(Heveatex Polymer N-1017)", at
    concentrations of 0, 1 or 5%, equivalent to 0, 750 or 3600 mg/kg
    bw/day, for 8 weeks. The daily intake of candelilla wax was
    approximately 0, 370 or 1800 mg/kg bw from the respective diets.
    Food and drinking water were provided  ad libitum. All rats were
    observed for signs of toxicity and mortality. Food and water intakes
    were measured twice weekly and body weights were recorded weekly. At
    the 4th week of treatment and at termination, blood and urine
    samples (procedure of collection not defined), from 4
    rats/sex/group, were taken and examined for haematology
    (haemoglobin, RBC, total and differential WBC) and urine parameters
    (transparency, odour, colour, pH, albumin and microscopic elements).
    At termination, all animals were sacrificed and autopsied. It was
    reported that no treatment-related adverse effects were observed in
    any of the investigated parameters (Harrisson, 1948).

         Groups of weanling Wistar rats (17 rats/sex/group), weighing
    50-60 g, were fed diets containing a mixture "(Product 17)" of
    styrene-butadiene polymer (50%) and candelilla wax (50%) at
    concentrations of 0, 1 or 5%, equal to 0, 680 or 3420 mg/kg bw/day
    for 27 weeks. Rats were observed for signs of toxicity and
    mortality. Food and drinking water intake and body weights were
    recorded weekly. Blood samples were collected (procedure not
    defined) from 5 rats/sex/group at the 13th and 25th week of
    treatment and examined for haemoglobin, RBC, total and differential
    WBC. On the same schedule, urine samples from 5 rats/sex/group were
    collected and examined for transparency, odour, colour, pH, albumin
    and microscopic elements. At termination, two-thirds of the animals
    from treated groups were sacrificed and autopsied. Rats of the
    control group were not sacrificed, but used as a control group in
    another one-year study. The following tissues and organs from 3
    males and 3 females, from each treated group, were microscopically
    examined: heart, lung, spleen, kidney, pancreas, small and large
    intestines, uterus, ovary, prostate, testicle and seminal vesicle.
    The author indicated in the results that, with the exception of a
    slight decrease of body weight gains in rats of both treated groups
    (predominantly after 7th week of treatment), the other investigated
    parameters did not indicate any toxic change attributable to the
    treatment (Harrisson, 1949).

         In four separate studies, 12 male and 12 female weanling albino
    rats (strain not identified) received a gum base containing 4.1 to
    6.1% of candelilla wax in the diet, at concentrations ranging from
    10 to 25%, for a period of 180 days (10% for the first 7 days, 20%
    for the following 7 days, and 25% for the remaining treatment
    period). During the entire treatment period, the daily consumption
    of candelilla wax was estimated to be 2400 mg/kg bw. Rats of the
    control group (12 males and 12 females) were fed a "colony" diet,
    free of the test material. All rats survived the treatment. Food
    intake and body weight gains (frequency of recording not defined)
    were comparable between treated and control rats. Urinalysis (number
    of animals used and frequency of collection not defined), gross and
    histopathology (number of animals examined not defined) revealed no
    changes that could be associated with the treatment (Hodge, 1973).

    2.2.2.2  Dogs

         Diets containing 0, 1 or 10% of a gum base containing 25%
    candelilla wax were fed to groups of young dogs (strain not defined,
    1-2 males and 2-3 females per group), for 6 months. Test dogs
    consumed approximately 0, 60 or 600 mg/kg bw/day candelilla wax from
    their diets. Dogs were housed individually and were fed twice daily
    at regular intervals (amount of food per feeding was not defined).
    They were observed daily and underwent careful physical examination
    every second week. Each dog was weighed regularly (frequency not

    defined). Blood and urine samples from an unstated number of dogs
    were collected (frequency not defined) and analyzed for unidentified
    parameters. At termination, 2-3 dogs from each treated group and 4
    dogs (sex not defined) from the control group were necropsied and
    the following tissues and organs were microscopically examined:
    skin, stomach, small and large intestines, liver, pancreas, kidney,
    urinary bladder, ovary, uterus, testicle, epididymis, prostate,
    lung, mesenteric lymph node, spleen, tonsil, bone marrow, heart,
    aorta, sciatic nerve, adrenal, thyroid and ribs (no individual
    animal data for histopathology were provided). It was reported that
    no treatment-related changes were observed (Harrisson, 1953).

    2.2.3  Long-term/carcinogenicity studies

    2.2.3.1  Mice

         Diets containing 0, 0.8 or 5.0% (equivalent to 0, 1200 or
    7500 mg/kg bw/day) of a mixture of gum base containing 25%
    candelilla wax, were fed to groups of 15 black agouti (C57) mice of
    each sex for 12 to 13 months. Mice were observed for signs of
    toxicity and survival. Body weights were recorded (frequency not
    defined). About 50% of the surviving mice/group were sacrificed at
    the 12th month of treatment, and remaining mice at the 13th month.
    It was not indicated which organs were examined or whether
    histopathology was performed. It was reported that the number of
    deaths in the 5% dose group exceeded those in the lower or control
    groups. The causes of death were not defined. The authors concluded
    that diets containing a gum base-25% candelilla wax mixture, at dose
    levels as high as 7500 mg/kg bw/day, were not carcinogenic in mice,
    under the conditions of this study (Hodge, 1973; FASEB, 1981).

    2.2.3.2  Rats

         Groups of Sprague-Dawley rats (30 rats/sex/group) were fed
    diets containing 0, 0.8, 2.0 or 5.0% mixture of either gum base 11
    or gum base 12 containing 25% candelilla wax for either 89 weeks
    (gum base 12) or 2 years (gum base 11). It was estimated that the
    actual intake of candelilla wax by rats was approximately 0, 125,
    300 or 750 mg/kg bw/day. Rats were housed individually, provided
    with food and drinking water  ad libitum and observed for signs of
    toxicity and mortality (frequency of observations not defined). Food
    intake and body weights were recorded weekly. Blood and urine
    samples from 2-6 rats/sex/group were collected at the 3rd, 6th, 12th
    and 23rd month of treatment and analyzed for unidentified
    parameters. One to seven rats/sex/group were sacrificed and
    autopsied 6 months, 1 year or 1 years after initiation of
    treatment. At termination, the following tissues and organs from 3
    males and 3 females (after 89 weeks) and 9 males and 9 females
    (after 2 years), of the appropriate group, were microscopically

    examined: lung, liver, spleen, kidney, adrenal, heart, pancreas,
    thyroid, stomach, small and large intestine, sciatic nerve, ovary,
    fallopian tube, uterus, testicle, seminal vesicle, prostate, bone
    marrow, eye, bone, skin, and salivary gland (no individual animal
    data for histopathology were provided). It was reported that no
    significant differences were observed in any of the investigated
    parameters, between animals of treated and control groups
    (Harrisson, 1953).

    2.2.4  Reproduction studies

         No standard reproduction study with candelilla wax has been
    conducted. The only available observations were incidental to the
    short-term study reported above (Harrisson, 1949) in which 3 male
    and 3 female rats were fed a diet containing a mixture of
    styrene-butadine polymer "(Product 17)" (50%) and candelilla wax
    (50%) at concentrations of 0, 1 or 5%, equivalent to 0, 680 or
    3420 mg/kg bw/day for 5 months prior to mating. Two of the three
    females from each dose level conceived on either the first or second
    mating, and produced normal litters (FASEB, 1981).

    2.2.5  Special study on mutagenicity

         Four Ames tests with  Saccharomyces cerevisiae, Escherichia
    coli and  Salmonella typhimurium showed that candelilla wax was not
    mutagenic, either with without the activation system (Table 1).

    2.3  Observations in humans

         No information available.

        Table 1. Results of mutagenicity assays on candelilla wax
                                                                                            

    Test System       Test object          Concentration      Results       Reference
                                           of candelilla
                                           wax
                                                                                            

    Ames test1        Saccharomyces        1, 25, 2.50        Negative      Brusick, 1976
                      cerevisiae D4        and 5.00%

    Ames test1        S. typhimurium       1, 25, 2.50        Negative      Brusick, 1976
                      TA1535,              and 5.00%
                      TA1537,
                      TA1538

    Ames test2        S. typhimurium       0.3-10 000         Negative      Mortelmans
                      TA1535,              g/plate                         & Eckford,
                      TA1537,                                               1979
                      TA1538, TA98,
                      TA100

    Ames test2        Echerichia coli      10-10 000          Negative      Mortelmans
                      WP2                  g/plate                         & Eckford,
                                                                            1979
                                                                                            

    1 Both with and without S-9 fraction of rat, mouse and monkey liver
    2 Both with and without S-9 fraction of rat liver
    
    3.  COMMENTS AND EVALUATION

         A number of older studies in mice, rats, and dogs were
    reviewed. A 6-month study in dogs and a 2-year study in rats, with
    the highest dietary levels equivalent to 600 and 750 mg/kg bw/day,
    respectively, showed no compound-related toxicity. These studies
    were considered to be of fundamental importance in the safety
    assessment. In addition, microbial tests for mutagenicity were
    negative.

         The Committee felt that the deficiencies noted in the
    individual studies, particularly in the light of current criteria,
    were counterbalanced to some extent by the consistent absence of
    adverse effects, and concluded that the present functional uses
    (glazing agent, component of chewing-gum base, surface-finishing
    agent, and carrier for flavour) did not raise any toxicological
    concerns.

    4.  REFERENCES

    ACT (1984). Final report on the safety assessment of candelilla wax,
    carnauba wax, Japan wax and beeswax.  J. Amer. Coll. Toxicol., 3:
    1-41.

    ASHRAF-KHORASSANI, M., & TAYLOR, L.T. (1990). Analysis of crude,
    purified, and synthetic candelilla wax using supercritical fluids.
     Liquid and Gas Chromatogr., 8: 314, 316, 318, 320.

    BRUSICK, D. (1976). Mutagenic evaluation of compound FDA 75-43,
    MX8006-44-8 candelilla wax, L.F. refined. Project No. 2468, Litton
    Bionetics, Inc.

    FASEB (1981). Evaluation of the health aspects of candelilla wax as
    a food ingredient. Federation of American Societies for Experimental
    Biology. Prepared for FDA, Contract No. FDA 223-78-210. Unpublished
    Report.

    FDA (1982). Candelilla wax, proposed affirmation of GRAS status as a
    direct human food ingredient.  Fed. Reg., 47: 35776-35777.

    HARRISSON, J.W.E. (1946). Product No. 12, type III GRS. Rubber and
    candelilla wax. Report No. 36731. LaWall and Harrisson, Consultants,
    Unpublished Report.

    HARRISSON, J.W.E. (1948). Product No. 16, 50/50 mixture candelilla
    wax and heveatex (Styrene and Butadiene) Polymer N-1017. Report
    38222. LaWall and Harrisson, Consultants. Unpublished Report.

    HARRISSON, J.W.E. (1949). Heveatex 50%, candelilla wax, hydrogenated
    fat. Report 39348. LaWall and Harrisson, Consultants. Unpublished
    Report.

    HARRISSON, J.W.E. (1953). Long-term safety studies. Resume of
    studies on young dogs and albino rats. LaWall and Harrisson,
    Consultants. Unpublished Report.

    HODGE, H.C. (1973). A critical review at the 1) Feeding studies of
    candelilla wax incorporated in gum base, 2) together with studies of
    the possible carcinogenic potential by other routes of
    administration. University of California, San Francisco.

    MORTELMANS, K.E. & ECKFORD, S.L. (1979). Microbial mutagenesis
    testing of substances compound report: F76-048, candelilla wax.
    Final Report, Project No. LSU-6909. SRI International.

    ROGERS, R. (1978). Monograph on candelilla wax. Submitted under:
    Contract No. FDA 223-76-2001. Informatic Inc., Maryland, USA.
    Unpublished report.


    See Also:
       Toxicological Abbreviations
       CANDELILLA WAX (JECFA Evaluation)