Alan J.
Collings, PhD
Metabolism of Cyclamate and Its
Conversion to Cyclohexylamine
Since cyclamates were first introduced in the early
1950s, arguments have raged over the potential
carcinogenicity of this artificial sweetener. Concern
over the safety of cyclamates arises from observations
that some individuals and experimental animals can
metabolize cyclamate to cyclohexylamine and that
cyclohexylamine has been shown to produce testicular
atrophy in experimental animals. This study examines
the absorption, excretion, and metabolism of cyclamate,
particularly its conversion to cyclohexylamine. In
addition, the potential toxicity and pharmacology of
cyclohexylamine are discussed. Diabetes Care 12:50-55,
1989
T
he artificial sweetener cyclamate, which is >30
times as sweet as sugar, was introduced as a food
additive in 1951 (1,2). In many ways it appeared
to be an ideal food additive, i.e., one that was
only slowly and partially absorbed and once absorbed
was rapidly excreted into the urine unchanged with
minimal toxic action. However, two observations radi-
cally altered the world's view on the safety of cyclamate.
The first was the observation by Kojima and Ichibagase
(3) that some people and certain animals could metab-
olize cyclamate to cyclohexylamine. The second was a
long-term study of rats involving the feeding of mixtures
of cyclamate, saccharin, and cyclohexylamine, which
caused an increased incidence of bladder tumors when
compared with controls (4). The latter observation led
to the banning of cyclamates as a food additive in many
From Allan J. Collings, PhD, Product Safety Consultant, 9 Arethusa Way, Bisley,
Surrey, England CU2-49B2, UK.
Send correspondence and reprint requests to Dr. Alan J. Collings at the above
address.
50
countries. Since then, arguments have raged over the
carcinogenicity of cyclamate. Ultimately, at the request
of the Food and Drug Administration (FDA), a National
Academy of Sciences National Research Council Com-
mittee reviewed the evidence on the carcinogenicity of
cyclamate, and in 1985 they issued their report (5). The
committee concluded that "the weight of experimental
and epidemiological evidence does not indicate that cy-
clamate by itself is carcinogenic."
This conclusion was based on the results of several
long-term studies on carcinogenicity, all of which proved
negative, despite the occasional positive result in certain
in vitro studies. In one study, N-nitrosomethyl urea was
implanted into the bladder of rats with some given cy-
clamate and others water. Those receiving cyclamate
showed a marked increase in the incidence of bladder
tumors (33). The committee recommended that the study
be repeated. They also recognized the problems asso-
ciated with the metabolism of cyclamate to cyclohex-
ylamine and saw the need to take the toxic action of
this metabolite into consideration in the overall evalu-
ation of cyclamate for widespread use (5). In addition,
the committee noted that there was suggestive evidence
from in vivo and in vitro studies in animals that cycla-
mate had cancer-promoting or cocarcinogenic activity
and, based on the epidemiological studies in humans,
that the use of cyclamate-saccharin mixtures may be
associated with a small increase in the risk of bladder can-
cer (5).
To appreciate the significance of the conversion pro-
cess, it is necessary to examine the absorption and ex-
cretion of both cyclamate and cyclohexylamine. So-
dium cyclamate given by subcutaneous injection is rapidly
excreted unchanged in the urine. In rats given a single
oral dose of sodium cyclamate, the cyclamate rapidly
appears in the urine and is followed by slow and steady
DIABETES CARE, VOL. 12, NO. 1, SUPPL. 1, JANUARY 1989

excretion over the next 40 h. Once cyclamate has been
absorbed into the body, it is rapidly excreted. The pro-
longed excretion pattern observed after oral administra-
tion suggests that absorption from the gastrointestinal
tract is slow and protracted (6). Cyclohexylamine given
orally is rapidly absorbed and excreted.
MATERIALS AND METHODS
The conversion of cyclamate to cyclohexylamine has
been observed in humans, rats, mice, and rabbits (2).
However, not all humans or rats have the ability to con-
vert. Studies have been undertaken in a number of spe-
cies, including humans, to establish the nature of the
conversion process. In one study, piglets with the ability
to convert cyclamate to cyclohexylamine were main-
tained on a powdered diet containing 0.1 % sodium cy-
clamate (10). After the ability to convert had been clearly
established, the animals were placed on a cyclamate-
free diet and given instead the same amount of cycla-
mate every day by subcutaneous injection as had been
ingested through diet.
The pattern of urinary cyclamate excretion was typi-
cally erratic in the first part of the study, but when cy-
clamate was injected subcutaneously, there was a mas-
sive increase in the amount of cyclamate excreted in the
urine. The change in the pattern of fecal cyclamate ex-
cretion was more dramatic. In the first part of the study,
fecal excretion was erratic, but when cyclamate was
given by injection rather than via the diet, the amount
of cyclamate in the feces steadily decreased to zero. On
return to cyclamate via diet, the fecal cyclamate levels
gradually increased.
On examination of urinary cyclohexylamine excre-
tion, the levels in the first part of the study were erratic,
but once the route of administration was changed, the
levels steadily decreased to zero. Once cyclamate was
added to the diet, the levels of urinary and fecal cy-
clohexylamine gradually increased to the levels ob-
served in the earlier part of the study. By combining
12 3 4 5 6 7 8 9 1 8 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 28 2 1 2 2 23 24 25 26
Tine Days
FIG. 1. Metabolism of cyclamate (NaCHS) in pigs.
DIABETES CARE, VOL. 12, NO. 1, SUPPL. 1, JANUARY 1989
A.). COLLINGS
468-
-NaCHS in diet NaCHS in diet-
368-
12 3 4 5 9 18 1112 13 1415 16 171819 28 21 22 23 24 25^6
FIG. 2. Sodium cyclamate (NaCHS) by subcutaneous in-
jection. CHA, cyclohexylamine.
urinary and fecal cyclohexylamine excretion, the overall
effect of changing the route of cyclamate administration
from oral to subcutaneous could be seen (Fig. 1).
Superimposing the graph of urinary cyclohexylamine
excretion onto that of fecal cyclamate excretion showed
that during the change from oral to subcutaneous
administration and back, the level of urinary cyclo-
hexylamine paralleled that of fecal cyclamate (Fig. 2).
From this it can be concluded that cyclamate must be
present in the gastrointestinal tract for conversion to cy-
clohexylamine.
In the second study, piglets were fed a powdered diet
containing 0.1 % sodium cyclamate throughout the study.
Urine and feces were collected daily and analyzed for
cyclamate and cyclohexylamine. The amount of cy-
clohexylamine excreted in the urine was erratic and var-
ied widely from day to day. When oral antibiotics were
given to the animals, the level of urinary and fecal cy-
clohexylamine rapidly decreased to almost zero (Fig. 3).
However, once antibiotic treatment had ceased, the abil-
ity to convert returned.
7 8 9 18111213141516171819 8212223 24 25 262728
Tine Days
FIG. 3. Metabolism of cyclamate in pigs [total cyclohex-
ylamine (CHA) excretion]. Pigs were fed 0.1% sodium cy-
clamate in diet.
51
METABOLISM OF CYCIAMATE
At the end of the study, these animals were returned
to a cyclamate-free diet until urine and feces were free
of both cyclamate and cyclohexylamine. After a sample
of the feces had been incubated with cyclamate, cy-
clohexylamine could be detected in the incubation mix-
ture. Further evidence of conversion as a microbiolog-
ical process can be seen in studies where nonconverter
rats were turned into converters.
Laboratory rats that were not converters were fed cy-
clamate for 6 mo with no conversion being observed.
When these animals were placed in the dirty cages va-
cated by converters, they acquired the ability to convert
within days (10). In humans and animals, cyclamate is
not totally absorbed from the gastrointestinal tract, re-
sulting in some cyclamate excretion in the feces.
In a study of four nonconverting human subjects, each
subject was given the same amount of sodium cycla-
mate daily for 14 days (7). The amount was taken in
divided doses between 0900 annd 2200. For the last
two 3-day periods, urine and feces were collected for
cyclamate analysis. The process was repeated three
times, each with a different dose of cyclamate. Inde-
pendent of the dosage, 40-70% of the cyclamate was
excreted in the urine (Table 1). With the slow and partial
absorption characteristics of cyclamate, there is ample
opportunity for metabolism to cyclohexylamine (7).
When a converter was given a daily dose of cycla-
mate, cyclamate was present in the urine within 24 h.
The urinary cyclamate levels followed an erratic course,
sometimes high, sometimes low. However, when the
ingestion of cyclamate ceased, the urinary levels de-
creased to zero over the next 3 days (8,9). In contrast,
cyclohexylamine did not appear in the urine until 24 h
after ingestion of cyclamate began. Cyclohexylamine
continued to be excreted for 5 days after cyclamate
ingestion had ceased.
The delay in the appearance of cyclohexylamine in
the urine after ingestion of cyclamate was due to the
amount of time taken for the cyclamate to reach the
conversion site. It is unlikely the delay was caused by
the induction of a metabolic process (Fig. 4). This view
is supported by the fact that the decay curve for cyclo-
hexylamine after the withdrawal of cyclamate was the
same shape as the buildup curve observed at the start
of the study. This phenomenon may explain why the
TABLE 1
Distribution of excreted cyclamate in urine and feces
Urinary cyclamate mg/dl
Urinary + fecal cyclamate
x 100
(mg/24 h)
Sodium cyclamate
intake (mg/24 h)
100 69 56 59
61 56 56
250
500 55 41 37
A-D, volunteers.
52
688
see
u 488-
* 3ee-
° 288-
v>
iee-
' 1 ' 2 ' 3 ' 4 ' 5 ' 6 ' 7 ' 8 ' 9 i e i l ' 1 2 ' 13' 14'15'16'17^18 19" 29'21'
Tine Days
FIG. 4. Urinary excretion of cyclamate (horizontally hatched
bars) and cyclohexylamine {diagonally hatched bars). Sub-
jects were given 1 g sodium cyclamate/day. No sample
was taken.
metabolism of cyclamate was not discovered in the ear-
lier studies, in which only a single dose was given. Once
conversion was found to occur, many studies were un-
dertaken to estimate the incidence of human conver-
sion. For these studies to be valid, it was necessary for
the subjects to have been given repeated daily doses of
cyclamate before having a 24-h urine sample taken for
cyclohexylamine analysis (6,11-17; Table 1).
RESULTS
Over 80% of the subjects studied converted <0.15% of
the ingested dose of cyclamate. Among the converters,
conversion levels ranged from slightly >0.15% to a few
people who showed conversion values of >60%. Both
adults and children had been assessed for the ability to
convert. In one study, 49 children between the ages of
3 and 18 yr were given 507 mg calcium cyclamate/day
for 4 days (16). A 24-h urine sample was collected and
analyzed for cyclohexylamine. Four of the children ex-
creted cyclohexylamine that was of a conversion level
up to 21.6%. How this could be considered "trace"
conversion by the National Research Council is difficult
to imagine (5).
In studies to determine the incidence of conversion,
different dosage regimens have been used (Table 2). In
a study that investigated this aspect of conversion, three
converters were maintained on a fixed daily dose of
cyclamate for 14 days, with total urine and feces col-
lected over the last two 3-day periods. The study was
repeated three times, each with a different daily dose of
cyclamate. As the dose of cyclamate was increased, the
proportion converted to cyclohexylamine decreased (7;
60
Fig. 5). When assessing the significance of this conver-
73
67 sion, the incidence and amount of conversion have to
be taken into consideration. However, some of the ap-
proaches taken have limited validity. One study took
DIABETES CARE, V O L . 12, N O . 1 , SUPPL. 1 , JANUARY 1989
 A.). COLLINGS

TABLE 2
Incidence of human converters

Percent dose converted to cyclohexylamine

Cyclamate intake n 0-0.15 0.15-1 1-20 20-60 >60 Ref.

0.25 g/day for 4 days 141 105 14 15 5 2 6

1-3 g/day for 30 days 11 4 3 3 1 11
1 g/day for 3 days 35 31 3 1 12
1 g/day for 17 days 5 4 1 13
5 g/day for 7-8 days 16 14 2 14
3 g/day for 17 or 18 days 3 1 1 1 15
3 g/day for 4, 9, or 14 days 93 85 3 2 3 16
0.5 g/day for 4 days 49 45 1 2 1 16
1 g/day for 28 days 10 4 3 3 17
Total
n 363 293 30 28 10 2
Percent 81 8 8 3 0.6

the conversion levels of all the subjects and worked out
the average conversion (31). The Food and Agriculture
Organization and the World Health Organization used
the degree of cyclamate absorption in a nonconverter
as a reference and then considered a proportion (30%)
of the unabsorbed cyclamate as available for conversion
(32).
In a nonconverter, 40-70% of ingested cyclamate was
excreted in the urine. However, cyclamate was slowly
and only partially absorbed. In high converters, the pro-
portion of ingested cyclamate excreted in the urine was
much lower than in nonconverters. This evidence strongly
suggests that significant conversion occurs as the cycla-
mate passes over potential gastrointestinal absorption
sites (7).
If cyclamate is ever reintroduced, it must be at a re-
duced level; therefore, the amount of metabolism of low
doses (higher conversion) has to be considered as the
worst case. The worst case is represented by those who
can convert >60% of ingested cyclamate. Although this
represents only 0.5% of the population, it includes many
people numerically.
Cyclohexylamine is an organic base with a pKa of
~9.2. This means that at its physiological pH, it is al-
most totally ionized and exists as the cyclohexylamine
ion. It is rapidly absorbed from the gastrointestinal tract
and excreted largely unchanged into the urine. Studies
have shown that further metabolism is possible (18,19),
although no further metabolism has been detected in
many studies (7,18).
In at least five 90-day feeding studies where cyclo-
hexylamine (as the hydrogen chloride salt) was incor-
porated into the diet, testicular atrophy, reduced body
weight gain, and hyperactivity were observed.
In feeding studies, rats were fed a powdered diet con-
taining 0.01, 0.05, 0.2, 0.5, and 1% cyclohexylamine
hydrochloride for 90 days. Body weight gain was re-
duced to the 0.2% dietary level in males and to 0.1%
in females. The weight of the testes postmortem was
reduced in the groups receiving 1.0 or 0.5% cyclo-
hexylamine (equivalent to 434 and 175 mg cyclohexy-
lamine • kg" 1 • day"1, respectively). Macroscopic ex-
amination of the testes showed that 13 of 15 males fed
1 % cyclohexylamine had small flabby testes with a bluish-
purple tinge, typical of testicular atrophy.
Histologically, in the 1% group there was total bilat-
eral degeneration of the testes (testicular atrophy) in 5
rats, 95% degeneration in 3 rats, ^70% degeneration
in 4 rats, 40% degeneration in 1 rat, and < 1 % degen-
eration in the remaining 2 rats. This indicates that the
effect of cyclohexylamine hydrochloride was severe in
13 out of 15 rats. Two of the rats in the 0.5% treatment
group showed a marked increase in the number of tub-
ules with evidence of early degeneration. The no-effect
level for testicular atrophy was ~100 mg • kg" 1 • day"1
(20-24).
288
M
188
c
z
u
9
n B c D
U o l u n t e e r
FIG. 5. Effect of sodium cyclamate dosage on conversion.
Hatched bars, 250 mg/day; striped bars, 500 mg/day;
checkered bars, 1000 mg/day.

DIABETES CARE, VOL. 12, NO. 1, SUPPL. 1, JANUARY 1989 53

METABOLISM OF CYCLAMATE
DISCUSSION
W
hen attempting to compare these results to
humans, not knowing the mechanism that
causes the testicular atrophy is frustrating. Ef-
forts have been made to investigate this
mechanism, but no satisfactory conclusion has been
reached. Because converters have been found among
children and adults, caution should be taken when con-
sidering the testicular toxicity of cyclohexylamine.
In the feeding studies, animals receiving cyclohex-
ylamine did not gain weight as fast as the control ani-
mals (20,23). In pair-fed studies, in which the same
amount of food consumed by the cyclohexylamine an-
imals was fed to the controls, the control animals gained
weight more rapidly than animals receiving cyclohex-
ylamine hydrochloride (20,23).
In another study, animals receiving cyclohexylamine
in their diet were more active than the control animals
(20). Whether this was due to the reduction of food
intake or to the toxic action of cyclohexylamine is not
known. The no-effect level for growth in the study was
50 mg • kg" 1 • day"1.
Cyclohexylamine is pharmacologically active, with an
action similar to reserpine (25-30). Administration of a
single dose by the oral or intravenous routes causes a
rise in blood pressure; in this instance, the mechanism
of action is the release of norepinephrine from the tis-
sues of the nervous system (30). It has been shown in
humans that a peak cyclohexylamine blood level of 0.5-
0.7 mg/L after the administration of a single oral dose
of cyclohexylamine is sufficient to cause a significant
rise in blood pressure. Cyclohexylamine blood levels of
this nature have been found in human converters who
ingest a regular amount of cyclamate, although no in-
crease in blood pressure was observed (7).
Whereas the acute pharmacological action of cyclo-
hexylamine has been examined, the effects of chronic
exposure have not. In one 90-day feeding study, blood
pressure was measured immediately before termination.
These animals did not show an increase in blood pres-
sure; in fact, blood pressure was markedly reduced (20).
Information from these studies precludes any assess-
ment of the results of conversion of cyclamate to cy-
clohexylamine in people who have cardiovascular dis-
ease or who are receiving chemotherapy involving the
cardiovascular or nervous system. The carcinogenicity
of cyclamate is thus far from being resolved.
When studies on the mechanism and conversion of
cyclamate are conducted, the following should be taken
into account: /) the extent of conversion of cyclamate
to cyclohexylamine—the worst case should be the point
of reference, 2) the effect of cyclamate on developing
and mature testes, 3) the effect of cyclohexylamine on
growth, and 4) the acute and chronic pharmacological
effects of cyclohexylamine and how it may affect various
illnesses and patients receiving chemotherapy. To date,
the debate has been dominated by the cancer-causing
54
issue, which has been largely resolved, except for the
problem of its possible cocarcinogenic activity. The
problem surrounding the metabolism of cyclamate to
cyclohexylamine still exists.
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