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References Anderson, R.L., Lefever, F.R. & Maurer, J.K. (1988). The effect of various saccharin forms on gastro-intestinal tract, urine and bladder of male rats. Fd. Chem. Toxic. 26: 665-669. Armstrong, B.K. (1985). Saccharin/Cyclamates: epidemiological evidence. In: interpretation of Negative Epidemiological Evidence for Carcinogenicity. International Agency for Research on Cancer, Scientific Publication No. 65, p. 129, IARC, Lyon. Arnold, D.L. (1983). Two-generation saccharin~ bioassays. Environmental Health Perspectives 50, 27-36. Arnold, D.L. & Boyes (1989). The toxicological effects of saccharin in short-term genotoxicity assays. Mutation Res. 221, 69-132. Arnold, D.L., Moodie, C.A., Grice, H.C., Charbonneau, S.M., Stavric, B., Collins, B.T., McGuire, P.F.,, Zawidzka, Z.Z. and Munro, I.C. (1980). Long-term toxicity of ortho- toluenesulfonamide and sodium saccharin in the rat. Toxicol. Appl. Pharmacol. 52: 13. Ashby, J. (1985). The genotoxicity of sodium saccharin and sodium chloride in relation to their cancer-promoting properties. Fd. Chem. Toxicol. 23: 507-519. Ashby, J. & Ishidate, M. Jr. (1986). Clastogenicity in vitro of the Na, K, Ca and Mg salts of saccharin; and of magnesium chloride; consideration of significance. Mutation Res. 163: 63-73. Ball, L.M., Renwick, A.G. & Williams, R.T. (1977). The fate of [1°C] saccharin in man, rat and rabbit and of 2-sulphamoyl [14C] benzoic acid in the rat. Xenobiotica 7, 189-203. Byard, J.L. & Goldberg, L. (1973). The metabolism of saccharin in laboratory animals. Fd. Cosmet. Toxicol. 11, 391-402. Carlborg, F.W. (1985). A cancer risk assessment for saccharin. Fd. Chem. Toxic. 23: 499- 506. Carlborg, F.W. (1985). A cancer risk assessment for saccharin. Fd. Chem. Toxic. 23: 499- 506. Chappel, C.I. (1992). A review and biological risk assessmenti of sodium saccharin. Regul. Toxicol'. Pharmacol. 15, 253-270. N Chowaniec, J. & Hicks, R.M. (1979). Response of the rat to saccharin with particular reference ~ to the urinary bladder. Br. J. Cancer 39, 355-375. w Cohen, S.M. (1985). Multi-stage carcinogenesis in the urinary bladder. Fd. Chem. Toxicol. ~ 23, 521-528. CA ~ ~ 10

Cohen, S.M., Ellwein, L.B., Okamura, T., Masui, T., Johansson, S.L., Smith, R.A. Wehner, J.M., Khachab, M., Chappel, C.L, Schoenig, G.P., Emerson, J.L. & Garland, E.M. (1991a). Comparative bladder tumor promoting, activity of sodium saccharin, sodium ascorbate, related acids, and calcium salts in rats. Cancer Res. 51: 1766-1777. Cohen, S.M., Cano, M., Garland, E.M., Earl, R.A. & Carson, S.D. (1991b). Role of silicates and protein in the proliferative effects of saccharin on the male rat urothelium. Carcinogenesis 12: 1551-1555. Eklund, S.H., Garland, E.M. & Cohen, S.M. (1992). Characterization of proteins of male rat urine which bind saccharin in vitro. Presented at 83rd American Association for Cancer Research Meeting, May 20-23, San Diego, CA. Elcock, M., & Morgan, R.W. (1993). Update on artificiali sweeteners and bladder cancer. Reg. Toxicol. and Pharmacol. In press. Ellwein, L.B. & Cohen, S.M. (1990). The health risks of saccharin revisited. Crit. Rev. in Toxicol. 20: 311-326. Fujita, J., Yoshida, 0., Yuasa, Y., Rhim, J.S.,, Hatanaka, M. & Aaronson, S.A. (1984). Ha~ ras oncogenes are activated' by somatic alterations in human urinary tract tumors. Nature (Lond.) 309: 464-466. Fujita, J., Srivastava, S.K., Kraus, M.H., Rhim, J.S.,,Tronick, S.R. & Aaronson, S:A. (1985). Frequency of molecular alterations affecting ras protoncogenes in human urinary tract tumors. Proc: Natl. Acad. Sci. USA 82: 3849-3853. Fukushima, S. & Cohen, S.M. (1980). Saccharin-induced hyperplasia of the rat urinary bladder. Cancer Res: 40: 734-736. Fukushima, S'., Shirai, T., Hirose, M. & Ito, N. (1986a). Significance of L-ascorbic acid and urinary electrolytes in promotion of rat bladder carcinogenesis. In: Diet, Nutrition and Cancer (Y. Hayashi et al., Eds.), pp. 159-168. Jpn. Sci. Soc. Press. Tokyo/VNU Sci. Press, Utrecht. Fukushima, S:, Thamavit, W., Kurata, Y. & Ito, N. (1986b). Sodium citrate: a promoter of bladder carcinogenesis. Jpn. J. Cancer Res. (GANN) 77: 1-4. Garland, E.M., Sakata, T., Fisher, M.J., Masui, T: & Cohen, S.M. (1989). Influences of diet and strain on the proliferative effect on the rat urinary bladder induced by sodium saccharin. Cancer Res. 49: 3789-3794. Garland, E:M., St. John4 M., Eklund, S.H., Mattson, B.J., Johnson, L.S., Cano, M. & Cohen, S.M. (1993). A comparison of the effects of sodium saccharin in NBR rats and in intact and', castrated male F344 rats. Cancer Research. In press. 11

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Sweatman, T.W. & Renwick, A.G. (1982). Tissue levels of saccharin in the rat during two- generation feeding studies. Toxicol. Appl. Pharmacol. 62, 465-473. Taylor, J.M., Weinberger, M.A. & Friedman, L. (1980). Chronic toxicity and carcinogenicity to the urinary bladder of sodium saccharin in the in utero-exposed rat. Toxicol. AppL Pharmacol. 54: 57-75. Tisdel, M.O., Nees, P.O., Harris, D.L. & Derse, P.H. (1974). Long-term feeding of saccharin in rats. In: Symposium: Sweeteners. (G.E. Inglett, Ed.), pp. 145-158. The AVI Publishing Company, Inc., Westport, Conn. Verma, A.K. Bryan, G.T. & Reznikoff, C.A. (1985). Tumor promoter 12-o-tetradecanoyl- phorbol-13-acetate receptors in normal human transitional epithelial cells. Carcinogenesis 6: 427-432. Weisburger, J.H. (1988). Carcinogenesis bioassays of natural and artificiat sweeteners. In: Sweeteners: Health Effects. pp. 193, 224. Princeton Scientific Publishing Company, Princeton. Williamson, D.S. & Cohen, S.M. (1989). Unpublished observations. 14

Table 1. Bladder Tumour Incidence in F,-Generation Male Rats in Two-Generation Bioassays of NaS FDA Study' WARF Study' CHPB Study' IRDC Study' Dietary Level # per % Group Incidence # per % Group Incidence # per % Group Incidence # per % Group Incidence 0 29 3.0 20 0 42 0 324 Ob 0.01 16 0 0.05 - - 20 0 0.1 27 0 0.5 - - 20 5.0 1.0 22 0 - - - - 658 0.8 3.0 - - - - - 472 1.7* 4.0 - - - - - - 189 6.3* 5.0 21 5.0 20 35* 45 26.9* 120 12.5* 6.25 - - - - - - 120 16.7* 7.5 23 30.0* - - - - 118 31.4* ' Food and Drug Administration, reported by Taylor et al. (1980); Wisconsin Alumni Research Foundation, reported by Tisdel g_al.. (1974); Canadian Health Protection Branch, reported by Arnold gt al. (1980a); International Research and Development Corp., reported by Schoenig gt al. (1985). ° The background incidence for total bladder neoplasia at IRDC is 0. 8% * Statistically significant zo9seEezoz

Bladder Hyperplasia (%) 30 20 -' 10-~ • • • r 4 Dietary NaS (%) Labelling Index 0.81 C + • r 6 + • + . 0.4~ + 0.0 Q.2~ • • 0 f-- T_ i 0 2 4 rosowcwOZ Dietary NaS (%) Bladder Tumors (%) 40, 30-{ 20 -J 10-I Light Transmittance (%) 1p0: + r B + • • 4 - I + • r 6 Dietary NaS (%) , T -T 2 4 Dietary NaS (~'o) 6 + • 8 8

Dietary NaS Prevented by Increased urine pH and sodium concentration NH4CI in the diet , -' - I,- or AIN, 76A diet Increased urine volume and decreased osmolality .4 High urinary protein levels in male vs female (au-globulin) Absent or less --~-• Urinary amorphous precipitate and crystals in female rats . Urothelial cytotoxicity V Absent or less --f-- • Urothelial hyperplasia and proliferation in female rats Bladder tumors Bladder tumor promotion~ in long-term bioassays in initiated rats

Legend for Figures Figure 1. Incidence of Urothelial Hyperplasia (A) and Bladder Tumors (B) in the IRDC Study of NaS in the Rat (Squire, 1985). Labelling Index Data (C) Taken from Cohen (1990), and Light Transmittance Findings (D) from a Recent Study (Cohen, Unpublished). Figure 2. Sequence of Urinary and Bladder Changes Observed in Male Rats Fed High Dietary Levels of NaS