Cancer Letters, 1 (1975) 21--24
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
A CHRONIC STUDY OF ARTIFICIAL SWEETENERS IN S Y R I A N GOLDEN HAMSTERS
J. ALTHOFF, A. CARDESA, P. POUR and P. SHUBIK The Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105 (U.S.A.)
(Received 23 June 1975)
SUMMARY Chronic administration of saccharin and sodium or calcium cyclamates in drinking water to Syrian golden hamsters, up to the maximum tolerated dose level, failed to induce an excess of tumors compared to controls; nor were any urinary bladder tumors found.
INTRODUCTION Experimental investigations of the artificial sweeteners cyclamate and saccharin have, in most cases, suggested that these substances are devoid of carcinogenic activity [3, 5, 6, 9, 10, 12, 14, 17], although a few experiments, in which high levels were fed led to a small incidence of bladder tumors [4, 8, 15]. Some positive results in these experiments may have been associated with urinary calculi formation or parasitization of the rat urinary bladder [ 2]. In this report, we describe findings following lifetime feeding of saccharin and calcium and sodium cyclamates to hamsters [1, 7, 13, 16]. MATERIALS AND METHODS Commercially available saccharin (Lot No. 91C-0370), made by the Maumee process, sodium cyclamate (Lot No. 121C-2480) (containing 10 ppm cyclohexylamine) and calcium cyclamate (Lot No. 82C-0730) (with a trace of cyclohexylamine) were used (Sigma Chemical Co., St. Louis, Missouri). The cyclamates were free from N-methylcyclohexylamine. Randomly bred Syrian golden hamsters of the Eppley colony, housed in groups of 5 in plastic cages b y sex, received Wayne pelleted diet (Allied Mills, Chicago, Illinois) and solutions of the test compounds or water (controls) ad libitum from 8 weeks of age. Fluid intake was measured weekly over a 24-h period. Gross.and histologic examinations were made of all hamsters. Urinary bladders were examined in step sections.
22 TABLE 1 LDso OF ARTIFICIAL SWEETENERS IN HAMSTERS BASED ON 8 DAYS' ADMINISTRATION IN DRINKING WATER; OBSERVED FOR 16 DAYS FROM BEGINNING OF TREATMENTS Compound
Sex No. of hamsters/group
Saccharin
F M Sodium cyclamate F M Calcium cyclamate F M
10 10 5 5 5 5
LD~0 (g/kg b.w.)
95% confidence limits (g/kg b.w.)
8.7 7.4 12.2 9.8 6.0 4.5
7.1--10.8 5.3--11.2 9.3--16.0 6.6--13.2 3.8--9.4 2.9--7.0
Observations Acute Toxicity. Because of the low toxicity of the artificial sweeteners, a single dose LD50 could not be determined. The LD50 of each compound [18] was calculated from mortality to day 16 in hamsters receiving levels of 0.625, 1.25, 2.5, 5.0 and 10.0 g/kg b.w. in water daily by stomach tube for 8 days (Table 1). Surviving animals were observed for life.
Subacute Toxicity. Test compounds were given in drinking water to 5 male and 5 female hamsters for 8 weeks at levels of 0.0, 0.625, 1.25, 2.5, 5.0 and 10%; the hamsters were subsequently kept for life. A m a x i m u m tolerated dose of saccharin, sodium or calcium cyclamate of 1.25% was selected for the chronic study, since animals showed no appreciable differences in weight from the control group at this level.
Chronic Toxicity. Levels of 0.0, 0.156, 0.312, 0.625 a n d 1.25% of each artificial sweetener were given in drinking water for life to groups of 30 male and 30 female hamsters. The average daily consumption varied from 353, 380 and 311 mg at the 1.25% level to 44, 47 and 38 mg at the 0.156% level for saccharin, sodium and calcium cyclamate, respectively. Average survival was from 50 ---60 weeks in all groups, except for those treated with 0.625 and 1.25% calcium cyclamate (43 and 29 weeks) and 1.25% sodium cyclamate (41 weeks). Generalized amyloidosis and vascular calcinosis, c o m m o n in animals over 40 weeks of age, were found in both untreated and treated hamsters. The incidence in all treated animals was higher than in contemporary controls, but did not significantly exceed that in other untreated hamsters of this colony. The following overall t u m o r incidences were obtained (with similar latency periods): 10.1% in 168 controls, 14.7% in 299 given saccharin, 14.2% in 247 given sodium cyclamate, and 11.9% in 210 given calcium cyclamate. The organ distribution and histologic types of neoplasms were within the range of spontaneously occurring tumors observed in these hamsters, Found were
23
adenomas of parathyroid, thyroid, bronchus, liver, endocrine and exocrine pancreas, adrenal cortex and kidneys; papillary polyps of trachea, gallbladder, colon and uterus; forestomach papillomas, pheochromocytomas of the adrenal medulla, hemangiomas of spleen and mesentery, leiomyofibromas of uterus, granulosa cell tumors of the ovaries, adenocarcinomas of thyroid, adrenal cortex and uterus; sarcomas of subcutaneous and mesenteric tissue and bone and malignant lymphomas. No urinary tract neoplasms were found. DISCUSSION
Certain chronic toxicity tests [4, 8, 15] have suggested the possible carcin~genicity of saccharin and cyclamates at high consumption levels in rats; however, many other experiments resulted in negative findings [3, 5, 6, 9 12, 14, 17]. The present experiment in Syrian golden hamsters adds to the volume of negative evidence on the carcinogenicity of saccharin and cyclamates. No urinary bladder neoplasms were observed, even though this species is considered by some authors to be a particularly good model for this type of neoplasm [1, 7, 13, 16] ; nor were any other neoplasms of a specific type associated with treatment in this study. All treated animals did display a degree of vascular calcinosis, which was somewhat higher than that of their contemporary controls; it was not, however, in excess of the incidence usually seen in the Eppley colony. ACKNOWLEDGMENTS
This study was supported by contracts PHS-43-68-959 and NO1 CP33278 from the National Cancer Institute, NIH, PHS. We thank Dr. L. Wallcave (Omaha) for the chemical analysis. REFERENCES 1 Althoff, J., Krfiger, F.W., Mohr, U. and Schm/ihl, D. (1971): Dibutylnitrosamine carcinogenesis in Syrian golden and Chinese hamsters. Proc. Soc. Exp. Biol. Med., 136, 168--173. 2 Clayson, D.B. (1974): Guest editorial - - bladder carcinogenesis in rats and mice: Possibility of artifcats. J. Natl. Cancer Inst., 52, 1685--1689. 3 Fitzhugh, O.G., Nelson, A.A. and Grawley, J.P. (1951): A comparison of the chronic toxicities of synthetic sweetening agents. J. Amer. Pharm. Assoc., 40, 583--586. 4 Friedman, L., Richardson, H.L., Richardson, M.E., Leftco, E.J., Wallace, W.C. and Sauro, F.M. (1972): Toxic response of rats to cyclamates in chow and semisynthetic diets. J. Natl. Cancer Inst., 49, 751--764. 5 Miyaji, T. (1973): Chronic toxicity of sodium saccharin and sodium cyclamate on rats: 28 months feeding. Internat. Syrup. Saccharin Res., Long Island, New York. 6 Munro, I.C., Moodie, C.A. and Grice, H.C. (1974): Assessment of the carcinogenicity of commercial saccharin. Annual meeting of the Society of Toxicology, Washington, D.C. 7 Oyasu, R., Kitajima, T., Hopp, M.L. and Sumie, H. (1972): Enhancement of urinary bladder tumorigenesis in hamsters by co-administration of 2-acetylaminofluorene and indole. Cancer Res., 32, 2027--2033.
24 8 Price, J.M., Biava, C.G., Oser, B . L , Vogin, E.E., Steinfeld, J. and Levy, H.L. (1970): Bladder tumors in rats fed cyclohexylamine or high doses of a mixture of cyclamate and saccharin. Science, 167, 1131--1132. 9 Roe, F.J.C., Levy, L.S. and Carter, R.L. (1970): Feeding studies on sodium cyclamate, saccharin and sucrose for carcinogenic and tumour-promoting activity. F o o d Cosmet. Toxicol., 8, 135--145. 10 Rudali, G., Goesy, E. and Muranyi-Kovaes, I. (1969): Recherche sur l'action canc~rog~ne g~ne du cyclamate de soude chez les souris. C.R. Acad. Sc. Paris, 269, 1910--1912. 11 Schm~hl, D. (1973): Fehlen einer kanzerogenen Wirkung yon Cyclamat, Cyclohexylamin und Saccharin bei Ratten. Arzneim.-Forsch. (Drug Res.), 23, 1466--1470. 12 Schm~/hl, D. and Kriiger, F.W. (1972): Fehlen einer synkarzinogenen Wirkung yon Cyclamat bei der Blasenkrebserzeugung mit Butyl-butanol-nitrosamin bei Ratten. Arzneim.-Forsch. (Drug Res.), 22, 999--1000. 13 So, B,T. and Wynder, E.L. (1972): Induction of hamster tumors of the urinary bladder by 3,2'-dimethyl-4-aminobiphenyl. J. Natl. Cancer Inst., 48, 1733--1738. 14 Taylor, J.D., Richards, R.K. and Wiegand, R.G. (1968): Toxicological studies with sodium cyclamate and saccharin. F o o d Cosmet. Toxicol°, 6, 313--327. 15 Taylor, J.M. and Friedman, L. (1974): Combined chronic feeding and three-generation reproduction study in the rat. Annual meetings, Society of Toxicology, Washington, D.C. 16 Tomatis, L., Della Porta, G. and Shubik, P. (1961): Urinary bladder and liver cell tumors induced in hamsters with o-aminoazotoluene. Cancer Res., 21, 1513--1517. 17 Verschuuren, H.G., Kroes, R., Peters, P. and Van Esch, J.G° (1973): Personal Communication. 18 Weil, C.S. (1952): Tables for convenient calculation of median effective dose (LD50 or ED50) and instructions in their use. Biometrics, 8, 249--263.
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
A CHRONIC STUDY OF ARTIFICIAL SWEETENERS IN S Y R I A N GOLDEN HAMSTERS
J. ALTHOFF, A. CARDESA, P. POUR and P. SHUBIK The Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105 (U.S.A.)
(Received 23 June 1975)
SUMMARY Chronic administration of saccharin and sodium or calcium cyclamates in drinking water to Syrian golden hamsters, up to the maximum tolerated dose level, failed to induce an excess of tumors compared to controls; nor were any urinary bladder tumors found.
INTRODUCTION Experimental investigations of the artificial sweeteners cyclamate and saccharin have, in most cases, suggested that these substances are devoid of carcinogenic activity [3, 5, 6, 9, 10, 12, 14, 17], although a few experiments, in which high levels were fed led to a small incidence of bladder tumors [4, 8, 15]. Some positive results in these experiments may have been associated with urinary calculi formation or parasitization of the rat urinary bladder [ 2]. In this report, we describe findings following lifetime feeding of saccharin and calcium and sodium cyclamates to hamsters [1, 7, 13, 16]. MATERIALS AND METHODS Commercially available saccharin (Lot No. 91C-0370), made by the Maumee process, sodium cyclamate (Lot No. 121C-2480) (containing 10 ppm cyclohexylamine) and calcium cyclamate (Lot No. 82C-0730) (with a trace of cyclohexylamine) were used (Sigma Chemical Co., St. Louis, Missouri). The cyclamates were free from N-methylcyclohexylamine. Randomly bred Syrian golden hamsters of the Eppley colony, housed in groups of 5 in plastic cages b y sex, received Wayne pelleted diet (Allied Mills, Chicago, Illinois) and solutions of the test compounds or water (controls) ad libitum from 8 weeks of age. Fluid intake was measured weekly over a 24-h period. Gross.and histologic examinations were made of all hamsters. Urinary bladders were examined in step sections.
22 TABLE 1 LDso OF ARTIFICIAL SWEETENERS IN HAMSTERS BASED ON 8 DAYS' ADMINISTRATION IN DRINKING WATER; OBSERVED FOR 16 DAYS FROM BEGINNING OF TREATMENTS Compound
Sex No. of hamsters/group
Saccharin
F M Sodium cyclamate F M Calcium cyclamate F M
10 10 5 5 5 5
LD~0 (g/kg b.w.)
95% confidence limits (g/kg b.w.)
8.7 7.4 12.2 9.8 6.0 4.5
7.1--10.8 5.3--11.2 9.3--16.0 6.6--13.2 3.8--9.4 2.9--7.0
Observations Acute Toxicity. Because of the low toxicity of the artificial sweeteners, a single dose LD50 could not be determined. The LD50 of each compound [18] was calculated from mortality to day 16 in hamsters receiving levels of 0.625, 1.25, 2.5, 5.0 and 10.0 g/kg b.w. in water daily by stomach tube for 8 days (Table 1). Surviving animals were observed for life.
Subacute Toxicity. Test compounds were given in drinking water to 5 male and 5 female hamsters for 8 weeks at levels of 0.0, 0.625, 1.25, 2.5, 5.0 and 10%; the hamsters were subsequently kept for life. A m a x i m u m tolerated dose of saccharin, sodium or calcium cyclamate of 1.25% was selected for the chronic study, since animals showed no appreciable differences in weight from the control group at this level.
Chronic Toxicity. Levels of 0.0, 0.156, 0.312, 0.625 a n d 1.25% of each artificial sweetener were given in drinking water for life to groups of 30 male and 30 female hamsters. The average daily consumption varied from 353, 380 and 311 mg at the 1.25% level to 44, 47 and 38 mg at the 0.156% level for saccharin, sodium and calcium cyclamate, respectively. Average survival was from 50 ---60 weeks in all groups, except for those treated with 0.625 and 1.25% calcium cyclamate (43 and 29 weeks) and 1.25% sodium cyclamate (41 weeks). Generalized amyloidosis and vascular calcinosis, c o m m o n in animals over 40 weeks of age, were found in both untreated and treated hamsters. The incidence in all treated animals was higher than in contemporary controls, but did not significantly exceed that in other untreated hamsters of this colony. The following overall t u m o r incidences were obtained (with similar latency periods): 10.1% in 168 controls, 14.7% in 299 given saccharin, 14.2% in 247 given sodium cyclamate, and 11.9% in 210 given calcium cyclamate. The organ distribution and histologic types of neoplasms were within the range of spontaneously occurring tumors observed in these hamsters, Found were
23
adenomas of parathyroid, thyroid, bronchus, liver, endocrine and exocrine pancreas, adrenal cortex and kidneys; papillary polyps of trachea, gallbladder, colon and uterus; forestomach papillomas, pheochromocytomas of the adrenal medulla, hemangiomas of spleen and mesentery, leiomyofibromas of uterus, granulosa cell tumors of the ovaries, adenocarcinomas of thyroid, adrenal cortex and uterus; sarcomas of subcutaneous and mesenteric tissue and bone and malignant lymphomas. No urinary tract neoplasms were found. DISCUSSION
Certain chronic toxicity tests [4, 8, 15] have suggested the possible carcin~genicity of saccharin and cyclamates at high consumption levels in rats; however, many other experiments resulted in negative findings [3, 5, 6, 9 12, 14, 17]. The present experiment in Syrian golden hamsters adds to the volume of negative evidence on the carcinogenicity of saccharin and cyclamates. No urinary bladder neoplasms were observed, even though this species is considered by some authors to be a particularly good model for this type of neoplasm [1, 7, 13, 16] ; nor were any other neoplasms of a specific type associated with treatment in this study. All treated animals did display a degree of vascular calcinosis, which was somewhat higher than that of their contemporary controls; it was not, however, in excess of the incidence usually seen in the Eppley colony. ACKNOWLEDGMENTS
This study was supported by contracts PHS-43-68-959 and NO1 CP33278 from the National Cancer Institute, NIH, PHS. We thank Dr. L. Wallcave (Omaha) for the chemical analysis. REFERENCES 1 Althoff, J., Krfiger, F.W., Mohr, U. and Schm/ihl, D. (1971): Dibutylnitrosamine carcinogenesis in Syrian golden and Chinese hamsters. Proc. Soc. Exp. Biol. Med., 136, 168--173. 2 Clayson, D.B. (1974): Guest editorial - - bladder carcinogenesis in rats and mice: Possibility of artifcats. J. Natl. Cancer Inst., 52, 1685--1689. 3 Fitzhugh, O.G., Nelson, A.A. and Grawley, J.P. (1951): A comparison of the chronic toxicities of synthetic sweetening agents. J. Amer. Pharm. Assoc., 40, 583--586. 4 Friedman, L., Richardson, H.L., Richardson, M.E., Leftco, E.J., Wallace, W.C. and Sauro, F.M. (1972): Toxic response of rats to cyclamates in chow and semisynthetic diets. J. Natl. Cancer Inst., 49, 751--764. 5 Miyaji, T. (1973): Chronic toxicity of sodium saccharin and sodium cyclamate on rats: 28 months feeding. Internat. Syrup. Saccharin Res., Long Island, New York. 6 Munro, I.C., Moodie, C.A. and Grice, H.C. (1974): Assessment of the carcinogenicity of commercial saccharin. Annual meeting of the Society of Toxicology, Washington, D.C. 7 Oyasu, R., Kitajima, T., Hopp, M.L. and Sumie, H. (1972): Enhancement of urinary bladder tumorigenesis in hamsters by co-administration of 2-acetylaminofluorene and indole. Cancer Res., 32, 2027--2033.
24 8 Price, J.M., Biava, C.G., Oser, B . L , Vogin, E.E., Steinfeld, J. and Levy, H.L. (1970): Bladder tumors in rats fed cyclohexylamine or high doses of a mixture of cyclamate and saccharin. Science, 167, 1131--1132. 9 Roe, F.J.C., Levy, L.S. and Carter, R.L. (1970): Feeding studies on sodium cyclamate, saccharin and sucrose for carcinogenic and tumour-promoting activity. F o o d Cosmet. Toxicol., 8, 135--145. 10 Rudali, G., Goesy, E. and Muranyi-Kovaes, I. (1969): Recherche sur l'action canc~rog~ne g~ne du cyclamate de soude chez les souris. C.R. Acad. Sc. Paris, 269, 1910--1912. 11 Schm~hl, D. (1973): Fehlen einer kanzerogenen Wirkung yon Cyclamat, Cyclohexylamin und Saccharin bei Ratten. Arzneim.-Forsch. (Drug Res.), 23, 1466--1470. 12 Schm~/hl, D. and Kriiger, F.W. (1972): Fehlen einer synkarzinogenen Wirkung yon Cyclamat bei der Blasenkrebserzeugung mit Butyl-butanol-nitrosamin bei Ratten. Arzneim.-Forsch. (Drug Res.), 22, 999--1000. 13 So, B,T. and Wynder, E.L. (1972): Induction of hamster tumors of the urinary bladder by 3,2'-dimethyl-4-aminobiphenyl. J. Natl. Cancer Inst., 48, 1733--1738. 14 Taylor, J.D., Richards, R.K. and Wiegand, R.G. (1968): Toxicological studies with sodium cyclamate and saccharin. F o o d Cosmet. Toxicol°, 6, 313--327. 15 Taylor, J.M. and Friedman, L. (1974): Combined chronic feeding and three-generation reproduction study in the rat. Annual meetings, Society of Toxicology, Washington, D.C. 16 Tomatis, L., Della Porta, G. and Shubik, P. (1961): Urinary bladder and liver cell tumors induced in hamsters with o-aminoazotoluene. Cancer Res., 21, 1513--1517. 17 Verschuuren, H.G., Kroes, R., Peters, P. and Van Esch, J.G° (1973): Personal Communication. 18 Weil, C.S. (1952): Tables for convenient calculation of median effective dose (LD50 or ED50) and instructions in their use. Biometrics, 8, 249--263.
