* Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892;
Clinical Pharmacology Group, University of Southampton, Biomedical Sciences Building, Bassett Crescent East, Southampton S016 7PX, United Kingdom;
Department of Pathology and Microbiology and the Eppley Institute for Research on Cancer and Allied Diseases, University of Nebraska, Omaha, Nebraska;
§ Nara Medical University, Kashihara, Nara 634, Japan;
¶ Covance Laboratories America, Inc., Vienna, Virginia 22182;
|| Office of the Director, National Cancer Institute, Bethesda, Maryland 20892.
Received February 23, 1999; accepted August 12, 1999
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ABSTRACT |
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Key Words: cyclamate; primates; carcinogen; toxicity; testicular abnormalities; malignancies; spermatogenesis.
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INTRODUCTION |
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Cyclamate is converted to the more toxic metabolite cyclohexylamine by the bacterial flora of the gastrointestinal tract (Renwick, 1986). In rats, testis is the organ most sensitive to the toxic effects of cyclohexylamine. Testicular atrophy, characterized by reduction in testicular weight and marked impairment of spermatogenesis has been reported in rats (Gaunt et al., 1974
and 1976; Oser et al., 1976
; Roberts et al., 1989
) and dogs (James et al., 1981
) but not mice (Roberts et al., 1989
). A comprehensive pair-feeding study has shown a no-observed-adverse-effect level (NOAEL) for testicular atrophy in rats at 100 mg/kg/day, with a clear dose-response relationship at 200 mg/kg/day and above (see Bopp et al., 1986
, for details). The clearance of cyclohexylamine is more rapid in mice than in rats (Roberts and Renwick, 1989
), which may explain why testicular toxicity has not been reported in mice (Bopp et al., 1986
). Testicular atrophy has also been reported following chronic administration of sodium cyclamate in the diet (mostly at 510%) to rats (Ferrando and Huchet, 1968
; Nees and Derse, 1965; Oser et al., 1975
). The atrophy was seen at the end of the chronic studies and associated with marked reductions in body weight and the weights of other organs. The effects in elderly rats could be due to metabolism of cyclamate to cyclohexylamine, but this would have required sustained and high levels of metabolism (> 10%; see Bopp et al., 1986
), which is unlikely in rat colonies (Bickel et al., 1974
; Oser et al., 1975
; Renwick, 1986
).
Cyclamate conversion to cyclohexylamine has also been studied extensively in humans (Bopp et al., 1986; Buss et al., 1992
; Collings, 1989
; Renwick, 1986
). There are major inter- and intraindividual variations in the extent to which the proportion of an oral dose of cyclamate that is not absorbed from the gastrointestinal tract is converted to cyclohexylamine by the intestinal flora (Renwick, 1986
). The ADI for cyclamate established by the JECFA (1982) and the SCF (1985) is based on the NOAEL for testicular atrophy produced by cyclohexylamine in rats with the application of a 100-fold safety factor and assuming 18% metabolism per day (see Renwick, 1986
).
This report presents results of a toxicity and carcinogenicity study of cyclamate in nonhuman primates that involved treatment for a period of up to 24 years, i.e., from 1970 to 1994.
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MATERIALS AND METHODS |
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In 1994, a decision was made to end the cyclamate study and euthanize the remaining 14 cyclamate and 16 control monkeys. Complete necropsies were carried out on all of the animals. The following organs were removed and fixed in buffered formalin solution: brain, pituitary, salivary glands, thyroid, thymus, tongue, cheek pouches, trachea, esophagus, lungs, heart, aorta, liver, gallbladder, pancreas, spleen, kidneys, adrenals, stomach, duodenum, small intestine, colon, urinary bladder, uterus, ovaries, testes, seminal vesicles, prostate, mammary tissue, lymph nodes (axillary, inguinal, hilar), skin, skeletal muscle, and bone (sternum). Samples of tissues were embedded in paraffin and histologic sections were stained with hematoxylin-eosin (H&E). Giemsa and PAS staining was also performed on the sections of the testes.
Plasma, urine, and testes sample analysis.
Samples of plasma, urine, and testes were collected, frozen, and transported to the University of Southhampton, U. K., where they were analyzed for cyclohexylamine using the method of Buss et al., 1992. The samples were analyzed in duplicate; samples showing poor replicate values (> 5% difference) were subject to further analyses to confirm the results. Triplicate standard curves of 35 different concentrations of cyclohexylamine spiked into the biologic matrix from control animals were analyzed with the samples; these were linear over the ranges studied (02 µg/ml for plasma; 010 µg/ml for urine; 010 µg/ml homogenate for testes) and showed intra-assay variability of 7% or less at each concentration. The sample data represent the means of 24 replicate values mostly within ± 5%.
The urine samples showed a very wide range of concentrations of cyclohexylamine, so that they had to be diluted to different extentsup to 1 in 100. In each case, 0.5 ml of the final dilution was used for analysis. The testicular samples were homogenized in 0.1 M phosphate buffer, and aliquots were extracted and analyzed. Samples from control monkeys showed a small interfering peak on HPLC that corresponded to low concentrations of cyclohexylamine (see Results).
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RESULTS |
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Testicular Findings
Evaluation of testicular function was carried out in 1982, after 12 years of dosing, in twelve cyclamate-treated monkeys (numbers 786J, 770J, 771J, 774J, 782J, 784J, 785J, 786J, 787J, 799J, 800J, 943M) and the age-matched controls. Semen analysis and measurements of serum testosterone and gonadotrophin levels did not reveal any differences between the cyclamate groups and the age-matched controls (data not shown). In addition, testicular biopsies from these monkeys showed normal histology. In 1990, the right testis was removed from one of the monkeys (943M) in the 100 mg/kg cyclamate group during a repair of an inguinal hernia. Microscopic examination of the testis did not show any abnormalities.
When the study was terminated in 1994, the surviving cyclamate-treated monkeys included ten males. With one exception (see below, monkey number 771J), the testicular size, color, and consistency of the cyclamate groups were not different from that of the age-matched controls. The testicular sections were examined independently by three pathologists. Focal spermatogenic arrest was seen in representative sections from several animals in both the cyclamate and control groups. In these cases, many areas showed normal spermatogenesis, and mature sperms were present in the epididymis. One monkey (771J) in the 100 mg/kg cyclamate group showed severe atrophy of the right testis, but a biopsy taken from the same testis in 1984 had shown normal spermatogenesis; the left testis had been removed from this monkey in 1990 during a repair of a long-existing inguinal hernia. Microscopic examination of the left testis showed areas of atrophic tubules and other areas showing normal spermatogenesis, and in view of the fact that this animal was in relatively poor health for many years due to chronic gastrointestinal problems, the testicular atrophy may not have been related to cyclamate exposure. Two monkeys (784J and 786J ) in the 500 mg/kg cyclamate group displayed focal germ cell aplasia (Sertoli-cell only tubules) mixed with areas showing normal spermatogenesis. Monkey number 784J also showed evidence of chronic orchitis. The consensus by the pathologists was that the testicular changes in the three cyclamate-treated monkeys listed above are probably not related to cyclamate exposure, as the remainder of the cyclamate group did not differ from the controls.
Other Histologic Findings
The pathologic findings of the two cyclamate groups and the control animals are shown in Tables 1, 2, and 3. Because sodium cyclamate has been implicated as a bladder carcinogen in rodents, special attention was given to macro- and microscopic examination of the urinary bladder. No premalignant lesions of the urothelium or bladder tumors were detected in either the cyclamate or control groups. Similarly, light and electron microscopic examination of the kidneys did not reveal any differences between the groups (data not shown). As shown in Tables 1, 2, and 3
, hyalinization of the pancreatic Langerhans islets was observed in two cyclamate-treated animals and three controls. This was associated with high blood glucose levels in one of the cyclamate-treated monkeys in the 500 mg/kg group (784J) and one of the control monkeys (678H). Hyalinization of the Langerhans islets has been observed sporadically in breeders and animals dosed with different test compounds in this monkey colony. The pathogenesis of this finding is unknown.
Cyclohexylamine Levels in Plasma, Urine, and Testes
Terminal cyclohexylamine levels were determined in plasma, urine, and testes, as described in the Materials and Methods section. Because of the presence of slight interference, the apparent cyclohexylamine concentrations in control monkey plasma ranged from 0.07 to 0.23 µg/ml respectively (Table 4). Given the terminal plasma concentrations in the controls, cyclamate monkeys 765J, 768J, 770J, and 795J were rated as nonconverters () (< 0.23 µg/ml); 771J, 782J, 790J, 791J, and 943M as low converters (+) (0.231.0 µg/ml); 772J and 773J as good converters (++) (15 µg/ml); and 786J, 787J, and 799J as high converters (+++) (> 5 µg/ml). The three high converters (which belonged to the 500 mg/kg group) had the highest testicular and urine cyclohexylamine levels (Table 4
). Interestingly, monkey 786J was one of two cyclamate-treated animals that showed focal germ cell aplasia (Sertoli-cell only tubules) of the testes.
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DISCUSSION |
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The increased incidence of bladder tumors was reported in rats fed with high dietary concentrations of a mixture of sodium cyclamate and sodium saccharin (Price et al., 1970) and led to the banning of cyclamate in the United States (U.S. Food & Drug Administration, 1970). Although cyclamate was associated with bladder tumors in another chronic feeding study (Oser et al., 1975
), the effect could not be reproduced in a number of other cyclamate carcinogenicity bioassays (Bopp, 1986). In contrast, chronic dosing with very high dietary concentrations of sodium saccharin and a variety of sodium salts consistently promote carcinogenesis in the rat bladder (Cohen et al., 1995
). However, a long-term feeding study of sodium saccharin showed no evidence of carcinogenic effect on the urinary tract in nonhuman primates (Takayama et al., 1998
). The lack of urothelial abnormalities in the saccharin and cyclamate-treated monkeys supports epidemiologic data that suggest that sodium cyclamate and sodium saccharin are not human bladder carcinogens (IARC, 1980; WHO, 1993
).
Three cases of different types of malignant tumors were found in the cyclamate monkeys. One case was a minute, well-differentiated prostate carcinoma that was detected at the time of necropsy of a cynomolgus monkey in the 100 mg/kg group. The other two cases (a colon carcinoma and a hepatocellular carcinoma) occurred in the 500 mg/kg group. Both monkeys presented with widely disseminated malignancies at 24 years of age. No malignant tumors were detected in the age-matched control group, however. We have previously reviewed the incidence of spontaneous tumor development in the breeders and control animals in this monkey colony since its onset in 1961 (Thorgeirsson et al., 1994). The spontaneous malignant tumor rate among 373 monkeys studied was 1.5% in cynomolgus, 2.8% in rhesus, and 8% in African green monkeys. Because statistical analysis could not be adequately carried out on the limited number of animals per group in this study, it is possible that a weak carcinogenic effect of cyclamate could be missed. However, the following findings would argue against cyclamate being carcinogenic: monkeys developed different types of malignant tumors, i.e., carcinomas of liver, colon, and prostate; tumors were detected after more than 20 years of dosing; tumors developed in both the 100 mg/kg and 500 mg/kg cyclamate groups; no proliferative or premalignant lesions were detected in the liver, colon, or prostate of the remaining cyclamate monkeys.
Cyclamate conversion to the toxic metabolite cyclohexylamines has been studied extensively in different experimental animal species as well as in humans (Bopp et al., 1986; Buss et al., 1992
; Collings, 1989
; Renwick, 1986
). The testis is the organ most sensitive to the toxicologic effects of cyclohexylamine in rats. A comparative study was carried out on the effects of cyclohexylamine in rats and cynomolgus monkeys and reviewed by the SCF (1995). A daily intake of 100 mg/kg of cyclohexylamine for 4 weeks produced testicular toxicity in the monkeys. However, 34 mg/kg (equivalent to approximately 200 mg/kg in rats, based on plasma concentration) for the same dosing period produced only slight changes in water intake and body weight but no histologic changes of the testes. The formation of cyclohexylamine was determined in our cyclamate-treated monkeys in 1984 (based on urine collections) and again at euthanasia in 1994. Three monkeys were categorized as high converters in 1994, with plasma cyclohexylamine levels of > 5 µg/ml, and these plasma levels are similar to those in rats showing adverse testicular histology (Roberts and Renwick, 1989
). Focal germ cell aplasia (Sertoli-cell only tubules) adjacent to histologically normal seminiferous tubules was observed in two of the 500 mg/kg monkeys (784J; 786J) but not in the control group. Monkey number 786J was a high converter in 1994; 784J was not assessed in 1994, but was a nonconverter in 1984. Sertoli-cell only tubules have also been reported in rats treated with cyclohexylamine (Bopp et al., 1986
; Creasy et al., 1990
). In considering the focality of these lesions and that they were observed in only two of the cyclamate monkeys, they are considered not to be induced by cyclamate per se, but a role for cyclohexylamine cannot be excluded. The three monkeys that were rated as high converters in 1994 were low or nonconverters when their cyclamate metabolism was determined in 1984; because of the limited number of observations and the fluctuation in cyclamate metabolism, it is impossible to estimate the level of long-term exposure of the monkeys to cyclohexylamine.
During the 24 years of dosing with cyclamate, there was no evidence of abnormal body weight changes. The only external malformation observed in the aged cyclamate monkeys was kyphosis of the thoracic vertebral column in three of the animals. Cyclohexylamine administration has been shown to increase blood pressure in humans (Eichelbaum et al., 1974), but cyclamate did not increase blood pressure, even in subjects with very high conversion to cyclohexylamine at the time of assessment (Buss et al., 1992
). This difference may be related to the time-dependent increase in the plasma concentrations of cyclohexylamine (Buss and Renwick, 1992
) following dosage with cyclohexylamine per se and cyclamate. The cyclamate monkeys in the present study did not show signs of heart problems and the necropsies did not reveal any specific cardiovascular abnormalities that were different from the controls.
In conclusion, the findings of this study showed that long-term feeding of nonhuman primates with high doses of cyclamate did not affect the general health of most of these animals. Clear evidence of compound-related testicular changes and tumors was not detected in the cyclamate monkeys after more than 20 years of dosing.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Bopp, B. A., Sonders, R. C., and Kesterson, J.W. (1986). Toxicological aspects of cyclamate and cyclohexylamine. CRC Crit. Rev. Toxicol. 16, 213306.[ISI]
Buss, N. E., and Renwick, A. G. (1992). Blood pressure changes and sympathetic function in rats given cyclohexylamine by intravenous infusion. Toxicol. Appl. Pharmacol. 115, 211215.[ISI][Medline]
Buss, N. E., Renwick, A. G., Donaldson, K. M., and George, C.F. (1992). The metabolism of cyclamate to cyclohexylamine and its cardiovascular consequences in human volunteers. Toxicol. Appl. Pharmacol. 115, 199210.[ISI][Medline]
Cohen, S. M., Cano, M., Garland, E. M., St. John, M., and Arnold, L. (1995). Urinary and urothelial effects of sodium salts in male rats. Carcinogenesis 16, 348358.
Collings, A. J. (1989). Metabolism of cyclamate and its conversion to cyclohexylamine. Diabetes Care 12, 5055.[Abstract]
Creasy, D. M., Ford, G. R., and Gray, T. J. B. (1990). The morphogenesis of cyclohexylamine-induced testicular atrophy in the rat: in vivo and in vitro studies. Exp. Mol. Pathol. 52, 155169.[ISI][Medline]
Eichelbaum, M., Hengstmann, J. H., Rost, H. D., Brecht, T., and Dengler, H. J. (1974). Pharmacokinetics, cardiovascular and metabolic actions of cyclohexylamine in man. Arch. Toxicol. 31, 243263.
Ferrando, M. R., and Huchet, B. (1968). Study of possible activity of sodium cyclamate on the rat in the course of three generations. Bull. Acad. Natl. Med. 153, 36.
Gaunt, I. F., Hardy, J., Grasso, P., Gangolli, S. D., and Butterworth, K. R. (1976). Long-term toxicity of cyclohexylamine hydrochloride in the rat. Food Cosmet. Toxicol. 14, 255267.[ISI][Medline]
Gaunt, I. F., Sharratt, M., Grasso, P., Lansdown, A. B. G., and Gangolli, S. D. (1974). Short-term toxicity of cyclohexylamine hydrochloride in the rat. Food Cosmet. Toxicol. 12, 609624.[ISI][Medline]
IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. (1980). Some non-nutritive sweetening agents. IARC 22, 55109.
James, R. W., Heywood, R., and Crook, D. (1981). Testicular responses of rats and dogs to cyclohexylamine overdosage. Food Cosmet. Toxicol. 19, 291296.[ISI][Medline]
JECFA (1982). Toxicological evaluation of certain food additives. Twenty-sixth report of the Joint FAO/WHO Expert Committee in Food Additives, Geneva, WHO Food Additive Series, No. 17, pp. 6677.
National Academy of Sciences, National Research Council, Committee on the Evaluation of Cyclamate for Carcinogenicity Report. (1985). Evaluation of Cyclamate for Carcinogenicity. National Academy Press, Washington, D.C.
Nees, P. O., and Serse, P. H. (1965). Feeding and reproduction of rats fed calcium cyclamate. Nature (Lond.) 208, 8182.[ISI][Medline]
Oser, B. L., Carson, S., Cox, G. E., Vogin, E. E., and Sternberg, S. S. (1975). Chronic toxicity study of cyclamate: saccharin (10:1) in rats. Toxicology 4, 315330.[ISI][Medline]
Oser, B. L., Carson, S., Cox, G. E., Vogin, E. E., and Sternberg, S. S. (1976). Long-term and multigeneration toxicity studies with cyclohexylamine hydrochloride. Toxicology 6, 4765.[ISI][Medline]
Price, J. M., Biava, C. G., Oser, B. L., Vogin, E. E., Steinfeld, J., and Ley, H. L. (1970). Bladder tumors in rats fed cyclohexylamine or high doses of a mixture of cyclamate and saccharin. Science 167, 11311132.[ISI][Medline]
Renwick, A. G. (1986). The metabolism of intense sweeteners. Xenobiotica 16, 10571071.[ISI][Medline]
Report of the Temporary Committee for the Review of Data on Carcinogenicity of Cyclamate. (1976). Department of Health, Education, and Welfare, DHEW Publication No. (NIH) 771437.
Roberts, A., and Renwick, A. G. (1989). The pharmacokinetics and tissue concentrations of cyclohexylamine in rats and mice. Toxicol. Appl. Pharmacol. 98, 230242.[ISI][Medline]
Roberts, A., Renwick, A. G., Ford, G., Creasy, D. M., and Gaunt, I. (1989). The metabolism and testicular toxicity of cyclohexylamine in rats and mice during chronic dietary administration. Toxicol. Appl. Pharmacol. 98, 216229.[ISI][Medline]
SCF (1985). Reports of the Scientific Committee for Food (Sixteenth series) Commission of the European Communities. Brussels. Report EUR 10210 EN. pp. 710.
SCF (1995). Opinion on cyclonic acid and its salts. Annex 1, Document III, 5121/96.
Takayama, S., Sieber, S. M., Adamson, R. H., Thorgeirsson, U. P., Dalgard, D. W., Arnold, L. L., Cano, M., Eklund, S., and Cohen, S. M. (1998). Long-term feeding of sodium saccharin to nonhuman primates: implications for urinary tract cancer. J. Natl. Cancer Inst. 90, 1925.
Thorgeirsson, U. P., Dalgard, D. W., Reeves, J., and Adamson, R. H. (1994). Tumor incidence in a chemical carcinogenesis study of nonhuman primates. Regul. Toxicol. Pharmacol. 19, 130151.[ISI][Medline]
U. S. Food & Drug Administration. (1970). Revocations regarding cyclamate-containing products intended for drug use. Fed. Regist. 35, 1364413645.
WHO. Saccharin and Its Salts. (1993). Toxicological evaluation of certain food additives and contaminants. WHO Food Additive Series 32, 105133.