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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