Mutation Research, 39 (1976) 1--28 © Elsevier/North-Holland Biomedical Press
THE MUTAGENICITY OF CYCLAMATES AND THEIR METABOLITES
B.M. CATTANACH M.R.C. Radiobiology Unit, Harwell, Didcot, Oxon, 0 X l l ORD (England)
(Received February 12th, 1976) (Accepted June 15th, 1976)
Contents Introduction ................................................ Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxicity/teratogenicity ....................................... Carcinogenicity ........................................... Phage i n d u c t i o n c a p a c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B i o c h e m i c a l effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M u t a g e n i c i t y tests Plant cells in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M a m m a l i a n s o m a t i c cells in vitro; c y t o g e n e t i c d a m a g e . . . . . . . . . . . . . . M a m m a l i a n s o m a t i c cells in vitro; gene m u t a t i o n . . . . . . . . . . . . . . . . . . M a m m a l i a n s o m a t i c cells in vivo; c y t o g e n e t i c d a m a g e . . . . . . . . . . . . . . M a m m a l i a n s o m a t i c cells; h o s t - m e d i a t e d assay . . . . . . . . . . . . . . . . . . . . M a m m a l i a n germ cells; s p e r m a t o g o n i a . . . . . . . . . . . . . . . . . . . . . . . . . . M a m m a l i a n germ cells; d o m i n a n t lethal studies . . . . . . . . . . . . . . . . . . . D r o s o p h i l a tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M i c r o o r g a n i s m s in vitro and h o s t - m e d i a t e d assay . . . . . . . . . . . . . . . . . . S u m m a r y and c o n c l u s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement ........................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
..
1 2 4 5 5 5 8 8 14 14 16 17 20 22 23 24 25 26
Introduction The calcium and s o d i u m salts o f c y c l a m i c acid (calcium and s o d i u m cycloh e x y l s u l p h a m a t e ) , generally k n o w n as c y c l a m a t e ( s ) , are dietetic drugs w h i c h have f o u n d extensive use as artificial, non-calorific sweeteners in a wide variety o f f o o d s a n d beverages. In c o n t r a s t t o saccharin, w h i c h previously held the d o m i n a n t p o s i t i o n as a sugar s u b s t i t u t e , the c y c l a m a t e s d o n o t have a b i t t e r after-taste at n o r m a l c o n c e n t r a t i o n s and are resistant t o boiling u n d e r the acid
or alkaline conditions usually occurring in the preparation of foodstuffs. Studies at Abbot Laboratories have shown that they have only 30 times the sweetening activity of sugar compared with 400 times for saccharin. However, when mixed with saccharin the combined sweetness is greater than the summation of the sweetness of either component. A 10 : 1 combination (cyclamate : saccharin) is the one which has been most extensively used [65,77]. Throughout the 1960's the intake of cyclamates as artificial sweeteners increased dramatically. The U.S. Department of Agriculture reported that consumption in the U.S. in 1964 was twice that in 1963 and five times that in 1959 [57]. A 7-fold increase over 1963 was predicted for 1971 [84]. About 60% was consumed in soft drinks alone [65] and it has been estimated that through this source a person on an otherwise normal diet could have a daily intake of up to 1% cyclamates (1% of total food consumption by weight). Other sources could further increase the proportion of cyclamate consumed [13,58]. On the enactment of the Food Additives A m e n d m e n t in the U.S. in 1958, cyclamates were included in the list of food additives "generally recognised as safe" (GRAS) for human consumption. However, the U.S. Dept. of Health, Education and Welfare subsequently banned the use of cyclamates in October 1969 on the evidence that under extreme conditions of dosage and length of treatment the 10 : 1 cyclamate : saccharin combination caused bladder tumours in rats when fed in the diet. This stand was reversed very shortly thereafter to allow food and drink containing cyclamates to be sold without prescription, though labelled as drugs. Then, in 1970, this decision was again reversed on the grounds that the safe feeding levels of cyclamate to rats had been revised downwards [12]. Now, allowing for the customary 100-fold safety factor, the assessed m a x i m u m safe daily dose for humans was calculated to be 168 mg/day for a 75 kg adult. This has a sweetening power of only 5.1 g of sugar. Hence, not being effective as drugs, cyclamates were no longer permitted to be marketed as such. The U.S. decision to ban cyclamates has since been followed by m a n y other countries. The ban on cyclamates has prompted considerable investigation into their toxicological, teratogenic, carcinogenic and mutagenic properties. This paper is concerned only with the possible mutagenicity of cyclamates but, because correlations between mutagenicity and carcinogenicity and perhaps also teratogenicity and phage induction capacity are now indicated, these'aspects will also be briefly considered. Of particular importance in the case of cyclamates is their metabolism. Hence, their metabolic fate and toxicity will also be summarized. Metabolism Studies both with 3ss- and 14C-labelled cyclamate in rats, rabbits and dogs have shown that, following i.v. administration, it is rapidly distributed throughout the body [55,70,77]. However, most of the radioactivity was seen to disappear within a few hours, residual amounts remaining only in the organs of excretion, i.e. the gastro-intestinal tract and kidneys. Such studies have further demonstrated t h a t 98--99% of the excreted radioactivity, faecal and urinary, is excreted in the form of cyclamates [13,27]. Some metabolism does occur,
however. More recent studies have shown that the nitrogen--sulphur bond appears capable of cleavage and the major conversion product is cyclohexylamine (CHA). Several investigators have detected CHA in the urine of human volunteers given low oral doses of sodium cyclamate and found the metabolite to be recovered in amounts equivalent to 0.7% of the administered cyclamate [34,37, 83]. The metabolism to CHA has also been observed in dogs, rats, guinea-pigs and rabbits [28]. The amount of CHA recovered is very variable and can be modified (see review [28]). Studies in man, dogs and rats have shown that prior feeding with cyclamates increases the ability to metabolize the c o m p o u n d . The gut flora have been found to be responsible. Thus, CHA excretion follows oral administration b u t not parenteral administration in rats; rat non-excreters can be converted to excreters by being caged with them for 2 days; anti-biotic pre-treatment reduces cyclamate metabolism; and little or no degradation to CHA is found when cyclamate is incubated in vitro with various tissues. Degradation does, however, occur when gut flora from rats or dogs are added. Anaerobic spore-forming bacteria in large and small intestine are the effective organisms in man [23]. A significant observation in assessing the risks of cyclamate ingestion is that bacterial activation is enhanced in the absence of sugar. Cyclamate metabolism may therefore be higher in individuals on non-fattening diets [28]. CHA itself m a y be further metabolized. Although Wallace et al. [82] could find no other metabolite than CHA in rats dosed with cyclamate for 1 year, Goldberg et al. [23] and Kojima and Ichibagase [35] have detected several other c o m p o u n d s in human urine after daily ingestion. These include cyclohexanol and cyclohexanone and N-hydroxycyclohexylamine (N-OHCHA) all of which are metabolites of CHA (Fig. 1).
CH 2
CH 2 -
C H 2 ~ - ~ C H CH2
CH 2 NSO3Ncl
CH2
Sodium cyclamate
OH 2
CH 2
all2
CH2
N - hydPoxycYClOhexylam ine
CH2
OH2
CH2
CH 2
Cyclohexanol Fig. I. Sodium
CH2
CH 2 ~ ' ~
CH NH2
CH2
CH 2
Cyclohexylamine
CH2
CH 2
CFI2
CH2
Cyclohexanone
CH2
CH 2
CH 2
CH 2
Cyclohexene cyclamate
a n d s o m e o f its m e t a b o l i t e s .
Cyclamates may also, undergo degradation before consumption. Higuchi et al. [26] have obtained evidence of a breakdown to cyclohexene in canned foods due to the presence of nitrite. CHA formation also occurs on the hydrolysis of cyclamates by organic c o m p o u n d s [13,28] and Fazio et al. [19] have ascribed the CHA content of a range of food products to the hydrolysis of the cyclamate during processing, with some further increase occurring during storage. Mutagenicity studies have concentrated on CHA as the principal metabolite of cyclamate b u t N-OHCHA has also received attention since there is reason to believe N-hydroxylated amines may be more biologically active than the parent c o m p o u n d [54]. Toxicity/teratogenicity Sodium and calcium cyclamate are not acutely toxic [13]. Richards et al. [65] report the LDs0 doses of sodium cyclamate for rats and mice to be a b o u t 17 g/kg when administered orally and 6--7 g/kg by i.p. injection. The LDso'S for CHA in mice have been quoted as 100 and 200 mg/kg for i.p. injection [13]. In mutation experiments, Green et al. [25] noted several deaths in rats following i.p. injections of CHA given in two doses (200 + 100 mg/kg) 4 h apart and Takano and Suzuki [76] observed some degree of lethality in mice given CHA in daily oral doses of 100 mg/kg. Sub-acute treatments with either cyclamate (up to 10% of the diet of rats, 4 g/kg/day with dogs) or CHA (up to 150 mg/kg/day p.o. with rats) have been found to give few ill effects. No impairment of reproductive performance has been found in several long term studies with rats exposed to high doses of cyclamate or CHA [2,20,31,57,65,84] and this, of course, provides little reason to suspect that such mutagenic effects as dominant lethality are being induced. Bajusz [3] described the occurrence of myocardial lesions, coronary sclerosis, and soft tissue calcification following the exposure of female hamsters to low doses (0.2 g) of calcium cyclamate, though they found no such effects in rats. A c o m m o n p h e n o m e n o n noted in several species exposed to chronic cyclamate treatments is weight loss, and the growth rates of immature and foetal rats have been seen to be reduced. This has been attributed to a reduced food uptake and utilization. Soft-stooling or even diarrhoea may occur with higher chronic doses of cyclamate but this appears to be due only to an osmotic action within the bowel, reducing the normal dehydration of the faeces [64]. Cyclamate treatments of pregnant mice and rats with doses up to 250 mg/kg or as 5% of the diet have not been found to cause e m b r y o lethality [21,43,58, 84] although single treatments with 100--500 mg/kg have been reported to increase foetal death in pigs [59]. The c o m p o u n d is known to be capable of passing through the placental barrier [21,70,77] and is retained by the foetus longer than by the mother. CHA a~ doses up to 100 mg/kg/day have been reported to cause e m b r y o lethal effects in mice [76]. No such effect was observed in rats with doses up to 150 mg/kg, however [2]. The cyclamates and CHA may have teratogenic properties. Both compounds have been reported to cause a wide range of teratogenic effects in chicken em-
bryos [22,64] and although these results cannot readily be extrapolated to mammals. Lederer et al. [38] have reported the occurrence of eye abnormalities in rat embryos following the treatment of the pregnant female with cyclamate. Teratogenic effects have not been observed by other investigators [2,57,65,76]. Carcinogenicity Early and detailed studies designed to test the carcinogenicity of cyclamates yielded completely negative results. However, the discovery of CHA as the principal metabolite of cyclamate, coupled with the propensity of amines to induce bladder cancer, later prompted the investigation of the bladder pathology of cyclamate-treated animals. Bladder cancers were indeed found in rats receiving very high doses of the 10 : 1 cyclamate : saccharin mixture over long periods of time, and, most of the animals in which tumours were detected had been shown to convert cyclamate to CHA [63]. Confirmation that cyclamate and not saccharin was responsible was later obtained b y other investigators (F.C.T. review [28]). Not all experiments have yielded the same conclusions, however, and tests on CHA for bladder carcinogenicity appear to be unconvincing [1, 28]. The incidence of bladder tumours in rats has also been found to increase following the surgical implantation of cholesterol pellets containing cyclamate in the bladder [ 7 ] b u t there are varied opinions as to the interpretations that can be drawn from the experimental procedure. Saccharin, too, gave positive results with this technique [28]. Since cyclamate carcinogenicity is so specific, essentially being limited to one organ in one species and only being effective at extremely high doses, this property would not seem to be a good indicator for cyclamate mutagenicity.
Phage induction capacity In presenting a rapid plate test for evaluating phage induction capacity Mayer et al. [51] reported their findings for a number of chemicals, these including CHA and N-OHCHA. Negative results were obtained with both compounds, while positive results were obtained with other, proven mutagens.
Biochemical effects Carr et al. [8] investigated the biochemical effects of calcium cyclamate (100--750 pg/ml) and CHA (10--50 pg/ml) in cultured human lung cells. Enzyme activities were stimulated after 5 h of exposure to the cyclamate b u t subsequently returned to normal. After 24 h de novo DNA synthesis was reduced, as was de novo RNA synthesis after 48 h. The higher concentrations of CHA proved toxic but the lower concentrations caused the same effects as the cyclamate. The results were taken to mean that the c o m p o u n d s interfere with DNA and RNA synthesis b u t it was not clear whether the site of action was DNA or DNA-dependent RNA synthesis.
78
15
36
74
75
Ref.
Na c y c l .
f N Caa ccyyccll.. CHA
Na c y c l .
J
1
)
10 -2 M
10--500 pg/ml
100--1000 pg/ml
HAI
THE MUTAGENICITY OF CYCLAMATES AND THEIR METABOLITES
B.M. CATTANACH M.R.C. Radiobiology Unit, Harwell, Didcot, Oxon, 0 X l l ORD (England)
(Received February 12th, 1976) (Accepted June 15th, 1976)
Contents Introduction ................................................ Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxicity/teratogenicity ....................................... Carcinogenicity ........................................... Phage i n d u c t i o n c a p a c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B i o c h e m i c a l effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M u t a g e n i c i t y tests Plant cells in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M a m m a l i a n s o m a t i c cells in vitro; c y t o g e n e t i c d a m a g e . . . . . . . . . . . . . . M a m m a l i a n s o m a t i c cells in vitro; gene m u t a t i o n . . . . . . . . . . . . . . . . . . M a m m a l i a n s o m a t i c cells in vivo; c y t o g e n e t i c d a m a g e . . . . . . . . . . . . . . M a m m a l i a n s o m a t i c cells; h o s t - m e d i a t e d assay . . . . . . . . . . . . . . . . . . . . M a m m a l i a n germ cells; s p e r m a t o g o n i a . . . . . . . . . . . . . . . . . . . . . . . . . . M a m m a l i a n germ cells; d o m i n a n t lethal studies . . . . . . . . . . . . . . . . . . . D r o s o p h i l a tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M i c r o o r g a n i s m s in vitro and h o s t - m e d i a t e d assay . . . . . . . . . . . . . . . . . . S u m m a r y and c o n c l u s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement ........................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
..
1 2 4 5 5 5 8 8 14 14 16 17 20 22 23 24 25 26
Introduction The calcium and s o d i u m salts o f c y c l a m i c acid (calcium and s o d i u m cycloh e x y l s u l p h a m a t e ) , generally k n o w n as c y c l a m a t e ( s ) , are dietetic drugs w h i c h have f o u n d extensive use as artificial, non-calorific sweeteners in a wide variety o f f o o d s a n d beverages. In c o n t r a s t t o saccharin, w h i c h previously held the d o m i n a n t p o s i t i o n as a sugar s u b s t i t u t e , the c y c l a m a t e s d o n o t have a b i t t e r after-taste at n o r m a l c o n c e n t r a t i o n s and are resistant t o boiling u n d e r the acid
or alkaline conditions usually occurring in the preparation of foodstuffs. Studies at Abbot Laboratories have shown that they have only 30 times the sweetening activity of sugar compared with 400 times for saccharin. However, when mixed with saccharin the combined sweetness is greater than the summation of the sweetness of either component. A 10 : 1 combination (cyclamate : saccharin) is the one which has been most extensively used [65,77]. Throughout the 1960's the intake of cyclamates as artificial sweeteners increased dramatically. The U.S. Department of Agriculture reported that consumption in the U.S. in 1964 was twice that in 1963 and five times that in 1959 [57]. A 7-fold increase over 1963 was predicted for 1971 [84]. About 60% was consumed in soft drinks alone [65] and it has been estimated that through this source a person on an otherwise normal diet could have a daily intake of up to 1% cyclamates (1% of total food consumption by weight). Other sources could further increase the proportion of cyclamate consumed [13,58]. On the enactment of the Food Additives A m e n d m e n t in the U.S. in 1958, cyclamates were included in the list of food additives "generally recognised as safe" (GRAS) for human consumption. However, the U.S. Dept. of Health, Education and Welfare subsequently banned the use of cyclamates in October 1969 on the evidence that under extreme conditions of dosage and length of treatment the 10 : 1 cyclamate : saccharin combination caused bladder tumours in rats when fed in the diet. This stand was reversed very shortly thereafter to allow food and drink containing cyclamates to be sold without prescription, though labelled as drugs. Then, in 1970, this decision was again reversed on the grounds that the safe feeding levels of cyclamate to rats had been revised downwards [12]. Now, allowing for the customary 100-fold safety factor, the assessed m a x i m u m safe daily dose for humans was calculated to be 168 mg/day for a 75 kg adult. This has a sweetening power of only 5.1 g of sugar. Hence, not being effective as drugs, cyclamates were no longer permitted to be marketed as such. The U.S. decision to ban cyclamates has since been followed by m a n y other countries. The ban on cyclamates has prompted considerable investigation into their toxicological, teratogenic, carcinogenic and mutagenic properties. This paper is concerned only with the possible mutagenicity of cyclamates but, because correlations between mutagenicity and carcinogenicity and perhaps also teratogenicity and phage induction capacity are now indicated, these'aspects will also be briefly considered. Of particular importance in the case of cyclamates is their metabolism. Hence, their metabolic fate and toxicity will also be summarized. Metabolism Studies both with 3ss- and 14C-labelled cyclamate in rats, rabbits and dogs have shown that, following i.v. administration, it is rapidly distributed throughout the body [55,70,77]. However, most of the radioactivity was seen to disappear within a few hours, residual amounts remaining only in the organs of excretion, i.e. the gastro-intestinal tract and kidneys. Such studies have further demonstrated t h a t 98--99% of the excreted radioactivity, faecal and urinary, is excreted in the form of cyclamates [13,27]. Some metabolism does occur,
however. More recent studies have shown that the nitrogen--sulphur bond appears capable of cleavage and the major conversion product is cyclohexylamine (CHA). Several investigators have detected CHA in the urine of human volunteers given low oral doses of sodium cyclamate and found the metabolite to be recovered in amounts equivalent to 0.7% of the administered cyclamate [34,37, 83]. The metabolism to CHA has also been observed in dogs, rats, guinea-pigs and rabbits [28]. The amount of CHA recovered is very variable and can be modified (see review [28]). Studies in man, dogs and rats have shown that prior feeding with cyclamates increases the ability to metabolize the c o m p o u n d . The gut flora have been found to be responsible. Thus, CHA excretion follows oral administration b u t not parenteral administration in rats; rat non-excreters can be converted to excreters by being caged with them for 2 days; anti-biotic pre-treatment reduces cyclamate metabolism; and little or no degradation to CHA is found when cyclamate is incubated in vitro with various tissues. Degradation does, however, occur when gut flora from rats or dogs are added. Anaerobic spore-forming bacteria in large and small intestine are the effective organisms in man [23]. A significant observation in assessing the risks of cyclamate ingestion is that bacterial activation is enhanced in the absence of sugar. Cyclamate metabolism may therefore be higher in individuals on non-fattening diets [28]. CHA itself m a y be further metabolized. Although Wallace et al. [82] could find no other metabolite than CHA in rats dosed with cyclamate for 1 year, Goldberg et al. [23] and Kojima and Ichibagase [35] have detected several other c o m p o u n d s in human urine after daily ingestion. These include cyclohexanol and cyclohexanone and N-hydroxycyclohexylamine (N-OHCHA) all of which are metabolites of CHA (Fig. 1).
CH 2
CH 2 -
C H 2 ~ - ~ C H CH2
CH 2 NSO3Ncl
CH2
Sodium cyclamate
OH 2
CH 2
all2
CH2
N - hydPoxycYClOhexylam ine
CH2
OH2
CH2
CH 2
Cyclohexanol Fig. I. Sodium
CH2
CH 2 ~ ' ~
CH NH2
CH2
CH 2
Cyclohexylamine
CH2
CH 2
CFI2
CH2
Cyclohexanone
CH2
CH 2
CH 2
CH 2
Cyclohexene cyclamate
a n d s o m e o f its m e t a b o l i t e s .
Cyclamates may also, undergo degradation before consumption. Higuchi et al. [26] have obtained evidence of a breakdown to cyclohexene in canned foods due to the presence of nitrite. CHA formation also occurs on the hydrolysis of cyclamates by organic c o m p o u n d s [13,28] and Fazio et al. [19] have ascribed the CHA content of a range of food products to the hydrolysis of the cyclamate during processing, with some further increase occurring during storage. Mutagenicity studies have concentrated on CHA as the principal metabolite of cyclamate b u t N-OHCHA has also received attention since there is reason to believe N-hydroxylated amines may be more biologically active than the parent c o m p o u n d [54]. Toxicity/teratogenicity Sodium and calcium cyclamate are not acutely toxic [13]. Richards et al. [65] report the LDs0 doses of sodium cyclamate for rats and mice to be a b o u t 17 g/kg when administered orally and 6--7 g/kg by i.p. injection. The LDso'S for CHA in mice have been quoted as 100 and 200 mg/kg for i.p. injection [13]. In mutation experiments, Green et al. [25] noted several deaths in rats following i.p. injections of CHA given in two doses (200 + 100 mg/kg) 4 h apart and Takano and Suzuki [76] observed some degree of lethality in mice given CHA in daily oral doses of 100 mg/kg. Sub-acute treatments with either cyclamate (up to 10% of the diet of rats, 4 g/kg/day with dogs) or CHA (up to 150 mg/kg/day p.o. with rats) have been found to give few ill effects. No impairment of reproductive performance has been found in several long term studies with rats exposed to high doses of cyclamate or CHA [2,20,31,57,65,84] and this, of course, provides little reason to suspect that such mutagenic effects as dominant lethality are being induced. Bajusz [3] described the occurrence of myocardial lesions, coronary sclerosis, and soft tissue calcification following the exposure of female hamsters to low doses (0.2 g) of calcium cyclamate, though they found no such effects in rats. A c o m m o n p h e n o m e n o n noted in several species exposed to chronic cyclamate treatments is weight loss, and the growth rates of immature and foetal rats have been seen to be reduced. This has been attributed to a reduced food uptake and utilization. Soft-stooling or even diarrhoea may occur with higher chronic doses of cyclamate but this appears to be due only to an osmotic action within the bowel, reducing the normal dehydration of the faeces [64]. Cyclamate treatments of pregnant mice and rats with doses up to 250 mg/kg or as 5% of the diet have not been found to cause e m b r y o lethality [21,43,58, 84] although single treatments with 100--500 mg/kg have been reported to increase foetal death in pigs [59]. The c o m p o u n d is known to be capable of passing through the placental barrier [21,70,77] and is retained by the foetus longer than by the mother. CHA a~ doses up to 100 mg/kg/day have been reported to cause e m b r y o lethal effects in mice [76]. No such effect was observed in rats with doses up to 150 mg/kg, however [2]. The cyclamates and CHA may have teratogenic properties. Both compounds have been reported to cause a wide range of teratogenic effects in chicken em-
bryos [22,64] and although these results cannot readily be extrapolated to mammals. Lederer et al. [38] have reported the occurrence of eye abnormalities in rat embryos following the treatment of the pregnant female with cyclamate. Teratogenic effects have not been observed by other investigators [2,57,65,76]. Carcinogenicity Early and detailed studies designed to test the carcinogenicity of cyclamates yielded completely negative results. However, the discovery of CHA as the principal metabolite of cyclamate, coupled with the propensity of amines to induce bladder cancer, later prompted the investigation of the bladder pathology of cyclamate-treated animals. Bladder cancers were indeed found in rats receiving very high doses of the 10 : 1 cyclamate : saccharin mixture over long periods of time, and, most of the animals in which tumours were detected had been shown to convert cyclamate to CHA [63]. Confirmation that cyclamate and not saccharin was responsible was later obtained b y other investigators (F.C.T. review [28]). Not all experiments have yielded the same conclusions, however, and tests on CHA for bladder carcinogenicity appear to be unconvincing [1, 28]. The incidence of bladder tumours in rats has also been found to increase following the surgical implantation of cholesterol pellets containing cyclamate in the bladder [ 7 ] b u t there are varied opinions as to the interpretations that can be drawn from the experimental procedure. Saccharin, too, gave positive results with this technique [28]. Since cyclamate carcinogenicity is so specific, essentially being limited to one organ in one species and only being effective at extremely high doses, this property would not seem to be a good indicator for cyclamate mutagenicity.
Phage induction capacity In presenting a rapid plate test for evaluating phage induction capacity Mayer et al. [51] reported their findings for a number of chemicals, these including CHA and N-OHCHA. Negative results were obtained with both compounds, while positive results were obtained with other, proven mutagens.
Biochemical effects Carr et al. [8] investigated the biochemical effects of calcium cyclamate (100--750 pg/ml) and CHA (10--50 pg/ml) in cultured human lung cells. Enzyme activities were stimulated after 5 h of exposure to the cyclamate b u t subsequently returned to normal. After 24 h de novo DNA synthesis was reduced, as was de novo RNA synthesis after 48 h. The higher concentrations of CHA proved toxic but the lower concentrations caused the same effects as the cyclamate. The results were taken to mean that the c o m p o u n d s interfere with DNA and RNA synthesis b u t it was not clear whether the site of action was DNA or DNA-dependent RNA synthesis.
78
15
36
74
75
Ref.
Na c y c l .
f N Caa ccyyccll.. CHA
Na c y c l .
J
1
)
10 -2 M
10--500 pg/ml
100--1000 pg/ml
HAI
