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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION



    SUMMARY OF TOXICOLOGICAL DATA OF CERTAIN FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES NO. 12






    The data contained in this document were examined by the
    Joint FAO/WHO Expert Committee on Food Additives*
    Geneva, 18-27 April 1977




    Food and Agriculture Organization of the United Nations
    World Health Organization



    * Twenty-first Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Geneva, 1977, WHO Technical Report Series No. 617

    SODIUM AND CALCIUM CYCLAMATES

         Sodium and calcium cyclamates were previously evaluated by the
    Joint FAO/WHO Expert Committee on Food Additives for the Fourteenth
    (FAO/WHO, 1970) and Eighteenth (FAO/WHO, 1974) Reports. Since the
    previous evaluation, additional data have become available.

    Feeding studies

         Kroes et al. (1975) designed a study to examine the short-term
    (reproductive, perinatal, teratogenic) and long-term (carcinogenic)
    toxicological effects of sodium cyclamate and acid saccharin, through
    6 generations of Swiss-SPF derived outbred mice. The following dietary
    treated groups were used:

    1. Control

    2. 5% sodium cyclamate

    3. 2% sodium cyclamate

    4. 5% sodium cyclamate + 0.5% acid saccharin

    5. 2% sodium cyclamate + 0.2% acid saccharin

    6. 0.5% acid saccharin

    7. 0.2% acid saccharin

         The P-generation consisted of 50 males and 50 females per group.
    Five weeks after the start of the study, 20 females and 10 males per
    group were selected to produce the F1 litter. The litter size was
    inadequate and the remaining 30 females were mated with 15 males to
    produce the F1a' generation. Subsequently, 20 females and 10 males
    were chosen to produce litters for each succeeding generation. The P,
    F3b and F6a generation consisted of 50 males and 50 females and were
    used to study carcinogenicity. The remaining generations consisted of
    10 males and 20 females. Reproduction was studied in generations
    F1a',, F2a, F3a, F4a and F5a. Perinatal studies were conducted
    with the F1a', F2b, F4b and F5b generations. A teratogenic study
    was undertaken with the F6b generation.

         Although the authors did not attempt to explain the F1a results,
    no consistent effect upon the various reproductive indices was
    observed due to treatment. Similarly no consistent toxic, embryotoxic,
    teratogenic or carcinogenic effects were observed. A total of 7
    bladder tumours were observed during the study in a total of 2100
    animals. The tumours were scattered among the different groups
    including controls.

    Testicular atrophy studies - Cyclamate

    Mouse

         Brantom et al. (1972) fed mice diets containing up to 7% sodium
    cyclamate and no testicular changes were observed.

    Rat

         In a 12-month feeding study with rats, Nees and Derse (1967) fed
    calcium cyclamate at dietary levels up to 10%. The absolute and
    relative testicular weights were similar to controls.
    Histopathological examination of the testes was not carried out.

         In a 3-generation reproduction study Ferrando and Huchet (1968)
    reported that calcium cyclamates at dietary levels of 3% caused
    atrophy of the testes. Following the administration of graded doses of
    a 10:1 cyclamate/saccharin mixture to rats for 24 months, Oser et al.
    (1970) found testicular atrophy in rats treated with 2500 mg/kg body
    weight/day. These animals also had significantly lower body weights
    than controls. Similar results were also reported by Taylor (1973)
    when calcium cyclamate was fed at a dietary level of 5% for 24 months.
    In another long-term study (28 months), Miyaji (1973) also found a
    marked testicular atrophy associated with a reduction in body weight
    of rats treated with sodium cyclamate or a 10:1 cyclamate/saccharin
    mixture at a dietary level of 5%.

         Friedman et al. (1972) fed rats diets containing up to 10% sodium
    or calcium cyclamate for 101 weeks and Schmahl (1973) fed 5% sodium
    cyclamate diets to rats for their entire lifespan. No testicular
    atrophy was observed in either study.

    METABOLISM

    Animal studies

         When various laboratory animals were fed diets containing
    cyclamate, there was generally an initial slow increase of
    cyclohcxylamine (CHA) excretion for the first 4 days after
    commencement of treatment. Thereafter, the urinary levels of CHA
    fluctuate markedly, often on a day-to-day basis. For example, Saunders
    and Wiegand (1967) reported that one rat fed a 5% cyclamate diet
    excreted a maximum amount of 81 mg of CHA in the urine on the 29th
    test day while on day 34 only 0.1 mg of CHA was excreted. Oser et al.
    (1968) classified rats as either high, low or zero converters of
    cyclamate to CHA, based on urinary levels of CHA collected during week
    27 of the study. Subsequent urinary samples from these animals
    collected 2.5 months later showed marked changes in cyclamate to CHA
    conversion. One animal previously classified as a zero converter was

    found to be a high converter. Similarly with pigs fed diets containing
    0.1% cyclamate, Collings (1971) found that high urinary CHA levels
    were often preceded by very low levels of CHA excretion, despite
    continued cyclamate administration.

         Cyclohexylamine has also been found in the urine of the dog,
    guinea-pig, rabbit and monkey following the administration of
    cyclamate (Renwick and Williams, 1972; Goldberg et al., 1969; Asahina
    et al., 1972; Ichibagase et al., 1972; Parekh et al., 1970).

    Clinical studies

         The metabolic conversion of cyclamate to cyclohexylamine has been
    extensively studied in over 1000 human subjects. The urinary excretion 
    of cyclohexylamine was found to be extremely variable from individual
    to individual and fluctuated greatly from day to day. The incidence of
    subjects that convert cyclamate to CHA would appear to be 10-30% of
    the test population. The majority of these convert < 0.1% to
    approximately 8% of the ingested cyclamate to CHA. A relatively few
    individuals convert up to 60% of the ingested cyclamate to CHA.

         Several studies indicate the conversion of cyclamate to CHA was
    inversely related to the cyclamate dose. When 5.0 g of cyclamate was
    ingested 0.6% of it was converted to CHA, while 13.4% of a 1 g dose of
    cyclamate was converted to CHA. For more specific details see Asahina
    et al., 1971, 1972; Collings, 1971; Davis et al., 1969; Golberg et
    al., 1969; Ichibagase et al., 1972; Leahy et al., 1966, 1967;
    Litchfield and Swan, 1971; Oser et al., 1968; Pawan, 1970; Saunders,
    1967, 1968; Saunders and Wiegand, 1967, 1968a, 1968b; Saunders et al.,
    1967; Renwick and Williams, 1972; Williams, 1971; Wills et al., 1968.

    CYCLOHEXYLAMINE

         Cyclohexylamine (CHA), a bacterial metabolite of cyclamate
    produced in the gastrointestinal tract, was previously evaluated by
    the Joint FAO/WHO Expert Committee on Food Additives for the
    Fourteenth (FAO/WHO, 1970) and Eighteenth (FAO/WHO, 1974) Reports.
    Since the previous evaluation, additional data have become available.

    Feeding studies

         Kroes et al. (1975) designed a study to examine the short-term
    (reproduction, perinatal, teratogenic) and long-term (carcinogenic)
    toxicological effects of cyclohexylamine through 6 generations using
    Swiss-SPF derived outbred mice. The dietary groups consisted of 0, or
    0.5% CHA.

         The P-generation consisted of 50 male and 50 female group. Five
    weeks after the start of the study, 20 females and 10 male group were
    selected to produce the F1a litter. The litter size was inadequate
    and the remaining 30 females were mated with 15 males to produce the
    F1a' generation. The P, F3b and F6a generations consisted of 50 males
    and 50 females and were used to investigate potential carcinogenicity.
    The remaining generation consisted of 10 males and 20 females.
    Reproduction was studied in generations F1a', F2a, F3a, F4a, F5a.
    Perinatal studies were conducted with the F1a', F2b, F4b and F5b
    generations and a teratogenicity study was undertaken with the F6b
    generation.

         The authors did not explain the F1a results. The CHA treatment
    resulted in a slight decrease in litter size, weight and survival of
    the pups in all generations. Maternal body weight and feed consumption
    were reduced due to CHA treatment and this may have contributed to the
    embryo-toxic effects.

         The perinatal study indicated that CHA significantly reduced the
    number of implantation sites in all but one generation. The
    teratogenicity study showed that CHA had a slight tendency to delay
    ossification.

         No bladder tumours were observed in the 300 CHA-treated animals,
    while one bladder tumour was observed in controls. The incidence of
    testicular atrophy in the CHA-treated males was similar to the
    incidence in control males.

    Testicular atrophy studies -Cyclohexylamine

    Mouse

         When CHA was administered at dietary levels of 0, 0.03, 0.1 or
    0.3% to mice for 80 weeks, no testicular changes were observed
    (Crampton, 1973).

    Rat

         Collings and Kirkby (1974) fed rats diets containing 0, 0.01,
    0.05, 0.1, 0.2, 0.5 or 1% CHA for 13 weeks. Decreased absolute
    testicular weights and body weight gains occurred at the two highest
    dosage levels. A dose-related decrement in food consumption occurred
    in the groups treated with 0.2, 0.5 and 1.0% CHA. A significantly
    increased incidence of histological changes in the testes (tubular
    degeneration) occurred only at the 1% dietary level. The no-effect
    level for testicular change was 0.5% (approv. 250 mg/kg bw) CHA in the
    diet.

         In a 13-week study (Gaunt et al., 1974) and a 24-month study
    (Crampton, 1973) in which CHA was fed at dietary levels of 0, 0.06,
    0.2 and 0.6% in the diet of rats, significantly decreased absolute
    testicular weights were observed in the 0.2 and 0.6% groups; these
    groups also exhibited significantly decreased body weight gains and
    food consumption. An increased incidence of testicular changes was
    observed with 0.6% CHA in both studies, with a questionable increase
    in the incidence at 0.2% CHA in the 13-week study. Five rats treated
    with 0.6% CHA in diet for 10 months exhibited normal fertility indices
    when tested for reproductive capacity. A no-effect level of 0.2%
    (approv. 100 mg/kg bw) was suggested.

         Crampton (1975) fed CHA at dietary levels of 0, 0.06, 0.1, 0.2
    and 0.6% to two strains of rats. The study included a pair-fed and a
    paired-weight control. In both strains, a significant increase in
    testicular effects (decreased spermatogenesis) was observed at 0.6%. A
    no-effect level of 0.2% ( 100 mg/kg bw) was suggested.

         CHA was administered to rats at dosage levels of 0, 15, 50, 100
    and 150 mg/kg body weight/day for 24 months (Oser et al., 1972).
    Trends towards dose-related decreases in food intake, body weight
    gain, absolute testes weight and relative testicular weights were
    observed, but testicular weights were not significantly different from
    controls. The histopathological changes in the testes from treated
    animals were not significantly different from controls.

         Calandra (1969) fed CHA for 24 months at dosage levels of 0,
    0.15, 1.5 and 15 mg/kg body weight/day to rats and reported that food
    intake, body weight gain, testes weights and testes histopathology of
    the treated animals were comparable to controls.

    Reproduction study

    Rat

         In a 5-generation reproduction study by Oser et al. (1972), CHA
    was fed at dietary levels of 0, 15, 50, 100 and 150 mg/kg bw/day. An
    increased number of resorption sites, lower number of pups cast alive
    and lower body weights of pups at weaning were found for the groups
    receiving 100 and 150 mg/kg. However, these effects were considered
    secondary, due to the decreased food consumption and decreased body
    weight gains of the pregnant dams in these groups.

    Calculation of an ADI

         The committee noted an unresolved problem in the toxicological
    evaluation of cyclamates due to its conversion to CHA by gut flora and
    the committee is also aware that additional studies concerning this
    problem are under way. CHA is known to produce testicular atrophy in
    rats. It was considered that this may be a species specific effect and
    it was not known whether this effect had ever occurred in human males.

         Data available from clinical studies of cyclamate metabolism
    indicates that about 40% of dietary cyclamate was absorbed and thus
    unavailable for conversion by gut flora into CHA. The inter- and
    intra-subject variability in the conversion of cyclamate to CHA was
    considered to be related to changes in the gut flora. It was estimated
    that for safety evaluation purposes an average figure of 30%
    conversion of the unabsorbed cyclamate could be accepted. Thus taking
    into consideration all these factors a temporary ADI was calculated as
    follows:

         No-effect level of cyclohexylamine (free base) in rat = 74 mg/kg
    bw/day. Temporary ADI (200-fold safety factor) for man for
    cyclohexylamine = 0.37 mg/kg bw.

    Converting cyclohexylamine to cyclamic acid

         Molecular weight cyclamic acid   = 2
         Molecular weight cyclohexylamine

         Thus if all cyclamate was converted to cyclohexylamine, the ADI
    for cyclamate would be 0.74 mg/kg bw.

         Since only 60% of ingested cyclamate is available for conversion,
    and only 30% of cyclamate is converted to cyclohexylamine, the
    temporary ADI for cyclamate is: 0.74  1.67  3.33 approv. 4 mg/kg
    bw.*

              
    *  Expressed as cyclamic acid.

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    See Also:
       Toxicological Abbreviations