FAO Nutrition Meetings
Report Series No. 48A
WHO/FOOD ADD/70.39
TOXICOLOGICAL EVALUATION OF SOME
EXTRACTION SOLVENTS AND CERTAIN
OTHER SUBSTANCES
The content of this document is the
result of the deliberations of the Joint
FAO/WHO Expert Committee on Food Additives
which met in Geneva, 24 June -2 July 19701
Food and Agriculture Organization of the United Nations
World Health Organization
1 Fourteenth report of the Joint FAO/WHO Expert Committee on Food
Additives, FAO Nutrition Meetings Report Series in press; Wld Hlth
Org. techn. Rep. Ser., in press.
CALCIUM AND SODIUM CYCLAMATE
Biological data
Biochemical aspects
The incorporation of inorganic S35 into protein polysacarchides
was used as a test system to determine the effects of sodium and
calcium cyclamate on membrane processes. Normally over 98% of S goes
into the macromolecule, less than 2% into S-containing amino-acids.
This was unaffected by the addition of cyclamate (Wortman et al.,
1968).
C14-labelled cyclamate was shown to have an average serum
half-life of 8 hours in dogs and rats. 32% of plasma cyclamate is not
protein bound. All tissues except the brain were shown to contain
cyclamate. Milk levels of cyclamate in lactating dogs were higher than
blood levels and amounted to 1% of the dose of cyclamate/1 milk
(Sonders & Wiegand, 1968).
C14-labelled orally given cyclamate was excreted in the urine
and faeces of rats after 3 days as follows: 20-30% appeared in the
urine, 70-80% in the faeces, 0% in the expired air. Urinary excretion
of C14-material was higher in converters due to the additional
excretion of C14-CHA (Lethco et al., 1969).
Sodium or calcium cyclamate fed to female Holtzman rats at a dose
level of 1 g/kg produced no hepatic microsomal enzyme induction as
measured by the N-demethylase, O-demethylase and phosphorothioate
detoxification activities. A 10:1 calcium cyclamate/calcium saccharin
mixture produced a small increase in activity attributable to the
saccharin (DuBois & Kinoshita, 1968).
Cyclohexylamine (CHA) conversion
Rat
Four groups of 35 male and 45 female rats were fed a 10:1 sodium
cyclamate/sodium saccharin mixture at 0, 500, 1120 and 2500 mg/kg
bodyweight per day for their lifespan. After 27 and 34 weeks
examination of the urines from each dose level showed a greater
percentage of converters at higher dose levels. Rats were either
non-converters (less than 0.1% of daily cyclamate ingested excreted
CHA in the urine), low converters (0.1-0.7% CHA of daily cyclamate
ingested excreted in the urine) or high converters (1% or more CHA of
daily cyclamate ingested excreted in the urine). Converting ability of
rats appeared to vary from time to time (Oser et al., 1968). Rats
pre-treated with water containing 0.5% calcium cyclamate were found to
develop the ability to convert cyclamate into CHA. Such pre-treated
rats were given 14C-labelled cyclamate I.P. and unlabelled cyclamate
orally. Gas-liquid chromatography and scintillation counting of the
urine at 24 hours showed that 0.9% of the cyclamate giver I.P. was
converted to CHA but about 38% of the cyclamate given orally was so
converted. About 90% of the injected cyclamate was excreted in the
urine at 24 hours compared with 58% of the cyclamate given orally.
When pre-treated rats were taken off the cyclamate containing diet the
ability to produce CHA from cyclamate fell to a low level (Renwick &
Williams, 1969), Groups of Holtzman rats were fed calcium cyclamate at
levels of 0 and 0.1% for 8 months. Twelve rats from each group were
then intubated with 14C-cyclamate following which urine and faeces
were collected for 5 days. None of the controls showed a conversion of
the labelled cyclamate to CHA, dicyclohexylamine or
hydroxycyclohexylamine. Seven of 11 cyclamate-fed rats converted
cyclamate to CHA, the labelled CHA accounting for 12 to 25% of the
total radioactivity seen in the urine. Traces of radioactive
dicyclohexylamine were seen in 2 of the urine samples (Prosky &
O'Dell, 1970).
The ability of rats to convert cyclamate to CHA can be
transferred to non-converter rats either by bodily contact with
converter rats for only 4 days or by contact with faeces from
converters (Unilever Research Labs, 1970). Groups of 7 male and 7
female rats were fed for 1 year on diets containing either sodium or
calcium cyclamate at 0, 0.4%, 2% and 10% levels. 24 rats acted as
controls. 53 oat of 53 rats became converters of which 25 excreted
less than 0.1% of ingested cyclamate as ChA, 16 excreted 0.1-1.0% of
ingested cyclamate as CHA and 12 excreted 2-38% of ingested cyclamate
as CHA. A G-ve bacterial red was isolated from the faeces of converter
rats that was capable of converting cyclamate to CHA in vitro but
lost this ability after several passages. Generally it was not
possible to obtain conversion of cyclamate to CHA by incubating
intestinal contents from various gut levels with cyclamate (Lethco et
al., 1969).
The contents of the hind gut of rats pre-treated with cyclamate
converted 0.8-3.5% of ingested cyclamate into CHA aerobically and
0.4-2.5% anaerobically. Liver, kidney, spleen and blood from these
rats failed to convert cyclamate to CHA (Renwick & Williams, 1969).
Feeding antibiotics, e.g. neomycin, to converter rats inhibited
the conversion of orally administered cyclamate to CHA. One rat which
converted 14% of oral cyclamate and 1% of i.v. cyclamate to CHA lost
this ability completely (Sonders, 1969).
Dog
Dogs given orally 100 mg/kg bodyweight/day were found to convert
up to 0.1% of the ingested cyclamate to CHA. The C14 of C14-labelled
cyclamate took longer to clear from the body after prolonged
consumption compared with clearance after a single dose. Incubation of
C14-cyclamate with large and small gut contents produced 0.13-0.25%
conversion of cyclamate to C14 CHA, possibly by Clostridium
perfringens (Goldberg et al., 1968; 1969).
Pig
Four pigs were shown to be able to convert orally fed cyclamate
to CHA. When neomycin was given to 2 pigs conversion was completely
inhibited. S.c. injection of cyclamate into 2 converter pigs for 8
days produced no CHA. No cyclamate appeared in the faeces and the
whole dose was excreted in the urine. Only anerobic incubation of
intestinal contents from the large colon produced CHA from cyclamate
hence this may be the probable site of conversion in the pig (Unilever
Research Labs., 1970).
Man
Various subjects given 512 mg/day of cyclamate orally converted
from 0-28% of the ingested cyclamate to CHA within 3-5 days after
starting consumption. Other metabolites identified were cyclohexanone,
cyclohexanol and N-hydroxy cyclohexylamine (Goldberg et al., 1968;
1969). 40 volunteers showed 5 converters at about 0.8% of the dose and
one subject converting 0.5% to CHA (Huntingdon Laboratories, 1967).
Forty-nine children, aged 3 to 10, consumed 516 mg of cyclamate per
day for 4 days. On the 4th day a 24-hour urine sample was collected
and assayed for cyclamate and CHA. 4 of the 49 children excreted 3.4
mg, 22 mg, 54 mg and trace amounts of CHA respectively. The incidence
of converters among the children was similar to that seen in 138
normal adults tested with cyclamate orally (Sanders et al., 1970).
Of 141 volunteers given 0.5 g sodium cyclamate per day for 3 days
some 25% converted more than 0.15% of the ingested cyclamate to CHA,
The maximum observed conversion rate was 60%. No CHA was detected in
100 people who did not ingest cyclamate. Converters given oral
cyclamate start excreting cyclamate in the urine within 24 hours but
CHA only after 24 hours. Peak excretion CHA occurs after 3-4 days. On
withdrawal of oral cyclamate there is rapid fall in urinary cyclamate
but CHA excretion does not fall until after 2 days. The percentage
converted had a biological half life of 20 minutes and that of CHA was
30 minutes. On. converter was used to demonstrate that CHA was not
necessary to start off conversion of cyclamate. While the absolute
amounts of urinary CHA excreted depend on the dose administered to
man, the proportion converted decreased with increased amounts
ingested. This was demonstrated in 3 volunteers. In 2 volunteers it
was shown that giving antibiotics stops conversion to CHA by
elimination of most of the intestinal bacterial flora (Unilever
Research Labs, 1970). 4 male and 3 female subjects received 3 g sodium
cyclamate in divided daily doses and 4 females received 1 g daily as a
single dose for 14-42 days. All converted cyclamate to CHA and
excreted on the average 2-8% of the ingested cyclamate as CHA in the
urine and faeces. CHA excretion became approximately constant for each
individual in the second week. A positive correlation was noted
between frequency of constipation and increases in CHA excretion.
Anaerobic incubation of cyclamate with faeces produced no CHA, all
cyclamate being recovered unchanged. One converter excreted 154 mg
CHA/day after ingestion of 1 g cyclamate and excreted 88% of the
ingested cyclamate in his faeces and urine (Davis et al., 1969).
Acute Toxicity
LD50
Animal Route. mg/kg Reference
Cyclamate-Saccharine Mouse oral 12800 Tremolières, 1964
(90%:10%)
" " " 21500 Mollet, 1966
Extemp Prep " i.p. 4600 Tremolières, 1964
Cyclamate-Saccharine " oral 17300 "
Tabs (Abbott)
Cyclamate-Saccharine " " >25000 "
Granules (Abbott)
Cyclamate-Saccharine Rat " >16500 "
(90%:10%) " i.p. 6500 "
Mouse
When the lipids in a standard diet were increased and
intraperitoneal injections of saccharin were given in increasing doses
to mice followed by a second injection 1 hour later of a fixed dose of
cyclamate a considerable lowering of the toxicity of both products was
noted (Tremolières, 1964).
5 groups of 5 male mice were given subcutaneous injections of
saccharin sodium, 0, 0.007, 0.07, 0.7 and 7 g/kg body-weight. A second
injection of 7 g/kg sodium cyclamate was given to all groups 1 hour
later. At first the mortality decreased gradually with increased doses
of saccharin to reach the lowest point with the proportion of 90%:10%
cyclamate/saccharin. After this the mortality increased sharply.
5 further groups of 5 male mice were given sodium cyclamate 0,
0.007, 0.07, 0.7 and 7 g/kg body-weight by subcutaneous injection. A
second injection of sodium saccharin 7 g/kg was given to all groups 1
hour later. Again the mortality was lowest with the proportion
cyclamate/saccharin 90%:10%. (Tremolières, 1964).
Special Studies
Six rabbits received sodium cyclamate at 0.5% and 2% in drinking
water for 4 weeks and the coagulation properties of their blood were
examined. At the 2% level was there a reduction in the Quick value and
factor VII. Another group of rabbits received 0.5% cyclamate in their
drinking water while on an anticoagulant. This addition increased the
anticoagulant effect (Gottinger et al., 1968). Tests to show the
effect of cyclamate excreted in the faeces on faecal moisture revealed
no correlation (Unilever Research Labs., 1970).
Cyclamate administration to guinea-pigs has been shown to produce
an allergic reaction of the delayed hypersensitivity type (Chung,
1969).
Oral cyclamate appears to have a diabetogenic action as evidenced
by blockage of the action of tolbutamide and chlorpropamide (Wisconsin
Alumni Research Foundation, 1969). In another experiment however
insulin has been shown to be more toxic in the presence of cyclamate
(Fels Research Laboratory, 1969).
Cyclamate has been shown to interfere with the intestinal
absorption of lincomycin by competing for plasma protein absorption
sites (Wagner, 1969). 14C-sodium, cyclamate infused into 2 rhesus
monkeys during the last trimester of pregnancy traversed the placenta
to the extent of about 29% while 14-CHA hydrochloride appeared to
diffuse freely across the placenta (Pitkin et al., 1969). Placental
transmission and foetal localization of radioactive cyclamate was
studied in 5 patients at the time of therapeutic abortion and
hysterectomy. Pregnancies were estimated to range from 68-93 days. A
solution in distilled water of 14C-cyclamic acid together with
nonradioactive sodium cyclamate to a total cyclamate concentration of
2 g/ml (0.105 mg/ml radioactive) was sterilized and infused into the
antecubital vein at constant rates. A volume of 50 ml was infused over
a 1 hour period in 3 cases, over a 3 minute period in one case and 99
ml were infused in 1 hour in one case. Maximum foetal blood values
were found to be approximately one quarter of comparable maternal
values. Amniotic levels were consistently low. Approximately 90% of
the radioactivity was recovered in the maternal urine over a 72 hour
period, 0.6% being recovered in the foetus in which it was widely
distributed (Pitkin et al., 1970).
Short-term studies
Rat
A litter mate paired feeding study in rats at dietary levels of
0, 0.5% 1% and 2% calcium cyclamate increased food consumption but
showed no adverse effects on growth, skeletal development or
gastrointestinal activity (Weinberg & Harrington, 1968).
Groups of 20 male and 20 female weanling rats were fed 0, 0.05%
and 0.5% sodium cyclamate for 3 months. No adverse effects were seen
in terms of growth, food intake, haematology, chemistry and gross
pathology (Loser, 1970).
2 groups of 6 rats were fed on diets containing 0 or 1%
cyclamate/saccharin (90:10%) mixture for 5 days, then subjected to
partial hepatectomy (90%) and post operatively maintained for 48 hours
on their respective diets. At autopsy on the 3rd day it was found that
hepatic regeneration was markedly diminished (15%) with the
cyclamate/saccharin mixture (Tremolières, 1964).
A group of 8 male and 8 female rats were given 1 mg/kg of a 10:1
cyclamate/saccharin mixture by gavage daily for 16 days. The control
group consisted of 2 male and 2 female rats. The growth rate was the
same in test and controls but the percentage weight gain was slightly
higher in the control groups (Mollet, 1966).
2 groups of 7 rats received 0 or 2% cyclamate/saccharin (90%:10%)
mixture for 5 weeks. Growth rate was only slightly lower in the test
group. In 3 test rats focal hepatic necrosis and congestive renal
lesions were found. (Tremolières, 1964).
2 groups 16 male and 16 female rats were given a 10:1
cyclamate/saccharin mixture in their diet at levels of 0.5% or 2%. The
control group consisted of 8 male and 8 female rats. After 16 weeks
50% of the animals in each group were sacrificed and the rest
continued on the same diet. Only 3 rats receiving the 0.5% level and 4
rats receiving the 2% level died during the experiment. No significant
effect of the test substance was noted on growth rate, food
consumption or haematology. Histological examination of the stomach,
liver, kidneys, gonads, spleen and pancreas revealed no alterations
(Mollet, 1966).
Guinea-pig
Two adult guinea-pigs received for 103-120 days in their drinking
water 0.5% sodium cyclamate, 2% (= 1200 mg/kg) sodium cyclamate or
0.3% sodium chloride. The water intake of the 2% test group was 30
ml/24 hours and the other 2 test groups were also kept at 30 ml fluid
intake/24 hours. Dead animals were replaced at once by fresh
guinea-pigs from stock. There was an apparent increase in mortality
and 20% decrease in body-weight at the 2% level. Histology of the
liver of 4 animals at both levels showed "reduced protoplasmic
content", vacuolization and occasional scattered focal necrosis. At
the 0.5% level there was less evidence of these hepatic lesions.
Histology of controls was apparently normal. The serum lactic
dehydrogenase and SGOT levels were raised at the 2% level but the
serum alkaline phosphatase level was normal. No changes were seen at
the 0.5% level (Gottinger et al., 1968). A continuation of this
experiment in female guinea-pigs kept for 64 days at 27, cyclamate in
their drinking water, 95 days at 0.5% cyclamate and 126 days at 0.3%
sodium chloride showed a loss in average body-weight of 36%, 37% and
23.4% respectively and a two-fold mortality in the 0.5% cyclamate
group compared with 0.3% saline (Hellauer, 1968).
Hamster
Oral administration of high doses of calcium cyclamate produced
myocardial degenerative lesions and coronary artery sclerosis when
compared with other calcium salts (Bajusz, 1969).
Human
30 adults took a 10:1 cyclamate/saccharin mixture (C:S) 6.4 g/day
for 12 months. Transient diarrhoea was noted in 5 subjects (Radding,
1967). For 6 weeks 32 adults took a C:S mixture 1.5 g/day increasing
to 7 g/day (7 days). At 7 g increased stool weight and softness was
noted (Olson, 1955). 184 children and adolescents took C:S mixture
1.0-1.5 g per 30 lb weight for 3 months and 6 months respectively,
There were some reports of soft light colour stools (Freese et al.,
1964). 20 adults with functional gastrointestinal conditions took 5
g/day C:S mixture for 10 weeks. Improvement in constipation was noted
(Batterman, 1966) 42 adult diabetics took C:S mixture 1.1 g/day for 6
months followed by 3.2 g/day for a further 6 months. No significant
changes in laboratory tests or physical status were noted (Stern,
1967).
Reproduction and teratogenicity studies
Mouse
Groups of 2-4 mice with control groups of 3 mice were given
single intragastric doses of sodium cyclamate at various stages of
pregnancy as follows: 62.5 mg on day 4-5, 125 mg on day 4-5 and 6-7,
250 mg on day 6-7 and 8-10 and 500 mg on day 6-7 and 8-10. The uteri
were removed on the eighteenth day, opened and the foetuses assessed
and absorption sites counted. No abnormalities were detected following
any administrations on days 8-10 but in all other instances the
majority of embryos had either been resorbed or appeared dead and
showed delayed development compared with controls. The foetal LD50
was calculated to be 180 mg/kg body-weight (Tanaka, 1964a; 1964b). In
embryo toxicity studies, groups of 10-20 mice were given single
intragastric doses of 5 and 10 g/kg of sodium cyclamate on days 5, 7
and 9 of pregnancy. The fetuses were delivered by caesarian section on
day 18. Foetal LD50 was found to be greater than 10.0 g/kg (Lorke,
1969). Groups of 10 mice were given intragastric doses of calcium
cyclamate of 86, 173, 346, 692 mg/kg sodium cyclamate of 62, 125, 250,
500 mg/kg and calcium cyclamate/sodium saccharin, 10:1 of 83, 166,
333, 667 mg/kg. Controls were 100 sham treated mice. Doses were given
on day 4, 6 and 9 of gestation. The foetuses were delivered by
caesarian section on day 18. Foetal swellings, implantation sites,
foetus numbers and viability were determined. Gross examination was
made for foetal growth and external evidence of terata, presence of
the latter being looked for additionally by serial sectioning and
clearing and alizerin staining. No cyclamate or cyclamate/saccharin
induced effects were seen (Industrial Bio-test Lab. Inc, 1967).
5 groups of 10 pregnant mice over 6 weeks were administered 0,
83, 166, 333 and 667 mg/kg body-weight of a 10:1 calcium
cyclamate/sodium saccharin mixture on gestation days 4, 6 and 9. All
animals were sacrificed on gestation day 18. The test material did not
significantly increase the incidence of foetal resorption and no
teratogenic effect could be related to treatment (Kennedy & Arnold,
1967).
Rat
20-23 female rats were dosed orally each day, from 6-15 day of
pregnancy, with the sodium cyclamate 50 mg/kg, 100 g/kg 250 g/kg.
These were compared with tap water (20 ml/kg) and saccharin and
sucrose as additional controls as well as 363 untreated controls. Dams
were autopsied on the 21st day of pregnancy. There were no significant
differences between any treated and control groups as regards litter
size, resorption rate and mean foetus eight. No abnormal incidence of
malformation was noted (Bein et al., 1967; Fritz. & Hess, 1968).
3 groups of 25 female rats were given by gavage a 10:1 sodium
cyclamate/ sodium saccharin mixture at levels of 0, 500, 2500
mg/kg/day based on their pre-pregnant weight from the 6th through the
16th day of gestation. No adverse responses were found regarding
behaviour, appearance, survival and reproduction. At 500 mg one dam
produced a malformed pup and another dam had 6 pups with absent lumbar
vertebrae. These changes are probably not attributable to the test
material as none occurred at 2500 mg/kg (Abbott, 1968).
Rabbit
Groups of 10 pregnant rabbits were given 50, 100 and 250 mg/kg of
sodium cyclamate or 5, 10 and 25 mg/kg of saccharin intragastrically
on days 6-18 of pregnancy. Pregnancies were terminated on day 29 by
caesarian section. Implantation sites, resorption sites and corpora
lutae were counted and foetuses were examined for evidence of visceral
or skeletal anomalies. No evidence of teratogenic or embryotoxic
effects were seen (Klotzche, 1969). Forty pregnant rabbits received
2500 mg/kg of sodium cyclamate/sodium saccharin (10:1) mixture by
gavage from days 6 through 18 of pregnancies. Thirty-eight pregnant
controls received water. Delivery was by caesarian section on day 29.
No effects attributable to the C:S mixture were seen in terms of
implantation and resorption sites live and dead foetuses, foetal
survival and terata (US Food and Drug Research Laboratories, 1969).
3 groups of 15 pregnant rabbits were given orally doses of 0, 500
and 2500 mg/kg of a 10:1 cyclamate/saccharin mixture once daily during
the 6th to 18th days of pregnancy. No teratogenic potential was noted.
One out of 61 pups from the high dosage level group showed
malformation of cranial structures, herniated abdomen and only one
kidney. The mean number of live pups in the treated groups in this
study was equal to or greater than that found in the control groups
(Oser, 1968).
Because of 1 malformed pup the experiment was repeated using 84
pregnant rabbits dosed intragastrically with 2500 mg/kg of 10:1
cyclamate/saccharin mixture once daily from 6th to 18th day of
pregnancy. No adverse effects were noted on the number of implantation
sites or live foetuses, resorption sites or dead foetuses in the test
group. No unusual malformations attributable to treatment were seen.
There was no evidence of teratogenesis (Oser, 1969).
Dog
Groups of 2 male and 4 female dogs were given a sodium cyclamate:
sodium saccharin (10:1) mixture (C:S) at 0, 0.5, - and 1.5 g/kg/day
for 2 years. No significant changes were seen in terms of growth, food
consumption, behaviour, appearance, haematology, biochemistry,
ophthalmological findings, endocrine function, hepatic biopsy,
reproductive performance, gross observation for terata and gross and
microscopic appearance of the major organs. A first litter obtained
from these animals and fed at similar levels for one year showed no
effects from the C:S mixture in terms of growth, food consumption,
haematology, biochemistry, endocrine function, ophthalmological
findings and gross and microscopic appearance of major organs. A
second litter was observed for periods ranging from 2 to 6 months
beyond weaning, receiving the C:S mixture at parental dosage levels.
In these animals growth, haematology, biochemistry, and gross and
microscopic findings were compared to control (Industrial Bio-test
Lab. Inc., 1968).
4 groups of 2 male and 4 female beagles were given 0, 0.5, 1 and
1.5 g/kg body-weight of a (10:1) sodium cyclamate:sodium saccharin
mixture for 2 years. The first and second litters received the same
dosage as their parents. No effect was noted in any generation on
blood chemistry, urine analysis or organ function. Reproduction
parameters including mating ability and gestation were normal; liver
biopsies, gross and microscopic pathology did not reveal any test
related lesions. There were no significant differences between test
and control animals in the first generation with regard to growth,
haematology, urology, organ function and pathology. The second litters
were also unaffected. No congenital malformations were observed in any
of the offspring (Jackson, 1968; Blockus et al., 1968).
Chick embryo
Calcium cyclamate and cyclamic acid were inoculated into fresh
fertile eggs either directly into the yolk or through the air cell at
0 and 96 hours of incubation. Decreased viability of embryos and a
high incidence of malformations were found at a dose level of 10 ppm
per egg when compared with sugar inoculation (Verrett, 1969). Similar
experiments using "doses equivalent to 18 g/day in man" produced
characteristic deformities in the embryos (Ghiani & Accame, 1969).
Special teratogenicity study
Rabbit
Rabbit embryos collected 30 hours post coitum in situ or in
vitro from superovulated mature doses were placed in a treatment
media containing a 10:1 sodium cyclamate:sodium saccharin mixture at
concentrations of 0, 3, 6 and 8 mg/ml. There was no effect on cleavage
in vitro or subsequent development of the same embryos in vivo
(Manor & Szymanski, 1968).
Mutagenicity studies
Cyclamate produced no chromosomal alterations in microbial cell
cultures and using a host-mediated assay with the S-typhimurium
cyclamate had no effect.
High levels of cyclamate have been found to cause breaks in
chromosomes of human leucocytes cultured in vitro (Stone et al.,
1969; Staltz et al., 1970).
Groups of 2 male and 2 female humans established as converters of
cyclamate to CHA and a similarly constituted group established as
nonconverters received 70 mg/kg of sodium cyclamate daily for 4 days.
A third control group received no cyclamate. Chromosome analyses were
performed on peripheral leucocyte cultures prior to and immediately
following the cyclamate ingestion. The frequency of chromosome
abnormalities in all 12 subjects prior to cyclamate was 0-4% with a
mean of 1.6% (based on counts of 100 metaphase cells per subject) and
after 4 days of treatment 0-4% with a mean of 1.8% (Abbott Labs.,
1970).
Long-term studies
Mouse
Sodium cyclamate was given in drinking water ad libitum to
groups of 30 day old mice. Cyclamate intake was estimated at 20-25
mg/mouse/day. Four mouse strains were used: C3H -40 males and 40
females, RIII -60 males, XVII/G -60 females, F1(C3H × RIII) -80
males. In each group half of the animals served as controls. Animals
dying prior to the appearance of a tumor in each group were not
considered in the evaluation. Average survival times for the animals
considered in the various groups ranged from 430 days to 667 days. In
the C3H and RIII strains no effects in terms of tumour numbers or
onset were seen. For the XVII/G strain tumor genic a greater number
multiple pulmonary tumours were seen. In the F1 (hybrid) strain the
hepatoma incidence was increased (Rudali et al., 1969).
Groups of 100 female Swiss mice were fed diets containing 0 and
5% sodium cyclamate for 18 months. At the start of the feeding 50 mice
in each group received intragastrically a single dose of 50 micrograms
of 3,4-benzopyrene in 0.2 ml of polyethylene glycol 400 (PEG) the
other 50 receiving PEG alone. Results did not indicate that the
feeding of cyclamate was associated with an increase in tumours either
arising in the G.I. tract or tumours at other sites, nor was there any
evidence of carcinogenicity or effects upon mortality (Roe et al.,
1970).
Implantation of cholesterol pellets containing 20% sodium
cyclamate into the urinary bladder have produced an increased number
of tumours in mice after 16 months. (Bryan & Ertürk, 1970).
4 groups of 35 male and 45 female rats were given a 10:1 sodium
cyclamate/sodium, saccharin mixture (C:S) at dosage levels of 0, 500,
1120 and 2500 mg/kg/day. At the 79th week each group was divided and
in addition to the C:S mixture the rats in 3 subgroups were dosed as
follows with cyclohexylamine until animals reached 2 years.
C:S Mixture Cyclohexylamine
500 mg/kg + 25 mg/kg
1120 mg/kg + 56 mg/kg
2500 mg/kg + 125 mg/kg
The other subgroups continued with their original C:S dosage. The
subgroups contained equal numbers of converters and non-converters.
The only physical sign seen in these test animals was occasional
soft stools or diarrhoea occurring primarily in male rats in the high
dosage group. Growth and food intake were similar in the control and
test animals. Haemograms and urine analyses performed at periodic
intervals revealed no adverse responses.
Bladder tumours were seen only at the 2500 mg/kg level between
the 78th and 105th week. Almost all, except for 1, occurred in male
converters. Out of 25 male controls 1 developed a papilloma. Out of 25
male test animals 2 developed papillomata and 5 developed carcinomata.
Out of 35 test females 1 developed a carcinoma (Price et al., 1969).
6 groups of male and female Osborne-Mendal rats were fed diets
containing 0.4%, 2% and 10% sodium or calcium cyclamates. Preliminary
histopathological evaluation of only the bladders of rats autopsied at
88 and 101 weeks indicate bladder effects in the animals fed cyclamate
as compared to controls. These effects were largely evident in the
animals autopsied at 88 weeks. They suggest a possible carcinogenic
effect, the calcium salt apparently being more potent. These findings
are tentative pending a complete evaluation (US Food and Drug
Administration, 1970).
Groups of 12 female and 6 male rats received 0, 0.8%, 1.6% and 3%
sodium cyclamate in their diet over 3 generations showed some
disturbance of oestrus and depression of growth as well as increased
mortality in the second generation and some testicular degeneration
(Ferrando et al., 1968).
Observations in man
Photoallergic dermatitis has been reported as a delayed reaction
to UV light in a patient taking 3600 mg calcium cyclamate per day. The
reaction was verified by testing with pure cyclamate and saccharin
separately (Lamberg, 1967). Pruritus, urticaria and other disorders
have been reported after ingestion of cyclamate (Feingold, 1968).
Groups of 8 men were given 0, 5, 10 and 18 g/day of sodium
cyclamate. Seven on the highest dose level develop a severe persistent
diarrhoea but the eighth tolerated this intake for 62 days. Some
persistent diarrhoea also occurred at 10 g/day in two men but none at
5 g/day (Wills et al., 1968).
In a continuation of studies previously described (Zollner &
Schnelle, 1969), patients suffering from liver and kidney disease were
continued on sodium cyclamate at dosage levels of 5 g/day and 2 g/day.
Patients receiving 2 g/day were observed over an average time of 21
months, those receiving 5 g/day were observed over 23 months in the
case of kidney patients and 30 months in the case of liver patients,
there being 10 of each type. Results of clinical biochemistry and
function tests did not in general show any deleterious effects on
renal or hepatic function nor were effects on the course of the
diseases seen (Zollner & Pieper, 1970). Epidemiologic study of the
incidence rates for bladder cancer indicates a gradual increase. While
epidemiological features of this neoplasm may be attributable to
cigarette smoking, an effect due to artificial sweeteners may be
undetectable because of the short observation time (Burbank &
Fraumeni, 1970).