NTP REPORT ON CARCINOGENS BACKGROUND
`
`DOCUMENT for SACCHARIN
`
`FINAL
`
`MARCH 1999
`
`
`Prepared for
`
`
`the October 30-31, 1997,
`
`Meeting of the Report on Carcinogens Subcommittee
`
`of the NTP Board of Scientific Counselors
`
`
`Prepared by
`
`
`Integrated Laboratory Systems
`
`Post Office Box 13501
`
`Research Triangle Park, North Carolina 27709
`
`NIEHS Contract No. N01-ES-25346
`
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`NTP Report on Carcinogens 1997 Background Document for Saccharin
`
`TABLE OF CONTENTS
`
`
`Proposed Report on Carcinogens De listing for Saccharin ..•...•..••••.•.•..••.••.••• 1
`
`Listing Criteria from the Report on Carcinogens, Eighth Edition ••••••••••••.•.• 7
`
`
`1.0 C~1\1DlC~ ~Rc=»~ER~~..•.......•.....•.•....•...•...•.......•....•.••.••.•..••.••.•.•...• ~
`
`1.1 Chemical Identification ..•.•.....•........•••......•••.••..•••.•.•...••••.•••••••.•••• 8
`
`1.2 Physical-Chemical Properties.•.•.•...•.•..•••.•.•..••••...•••••.••.••.••.•••••••••• 9
`
`
`•
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`2.0 HUMAN EXPc=»SU'RE ..•.•••.....•.•........••........•••.•...•.••......•.••••.•••••••..•••••.•••1 0
`
`2.1 Production •••.•.•.•••••..•••...••••.••••...•.•••.•••.•••.•••••••••.•••••••••.•••••••••••••••10
`
`Table 2-1 Forms of Saccharin Produced by PMC Specialties Group••••11
`
`2.2 Use .•••••••••••..•••••••.•••••..•••...••••..••••...•••..•••.•••..••••••.••••••.•••••••••••••••••12
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`2.3 Environmental Exposure ..........•........••.......•........•.......••..•...•••..... 13
`
`2.3.1 Environmental Releases........................................•........•13
`
`Table 2-2 Releases of Saccharin to the Environment•••••••.••••....13
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`2.3.2 Environmental Occurrence ..•••...•..•••.•.•..••••...••.••••..•••••••••13
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`2.3.3 Drinking Water and Food •.•.•••.•••...••.••••....•..•...•••..•.••.•••.•14
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`2.3.4 Consumer Products ••........•.......••........•.................•.......... 14
`
`Table 2-3 USDA Nationwide Food Consumption Survey
`
`(1977-1978): Total Calculated Saccharin Intake Levels•••14
`
`2.3.5 Biomarkers of Exposure.........................................••.......14
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`2.3.6 Occupational Exposure ....................................................15
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`Table 2-4 NIOSH National Occupational Exposure Survey
`
`(NOES, 1981-83): By Industry .•....•••.•.......••...••..•..••••••..••15
`
`2.4 Regulations .......•..........••.........••.......•••................•..•...••......•.......16
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`2.4.1 Occupational Exposure Limits ••...••.••••.•••••••••.••••••••••••••••••16
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`2.4.2 Other Standards and Criteria••••.•••.••••••.••••••••••.•••••.•••••••••16
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`3.0 HUMAN ST'UDIES••••••.•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••19
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`3.1 IARC (1980) Review of Saccharin Epidemiology •.••.•..••••..••••••••.•••20
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`3.2 Human Studies Published Post IARC (1980) .•.••.•..•..•••••••..••••...•.•21
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`3.2.1 U.S. Case-Control Studies .••..•••.•....•.•.•....••••.•...•••.....••.....21
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`3.2.2 Canadian Case-Control Studies •.•.•.••••••..••••.••••••.••••••.••••••23
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`3.2.3 Case-Control Studies From Other Countries ••••••.•••••••••••23
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`3.2.4 Descriptive Studies .•.•.•......••.............•.•.•.....•••....•.......•....24
`
`3.2.5 Meta-Analysis••.•••••••••••..•••••••••.•••••.••••••..••••••.••.••.••.••••.•..•24
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`NTP Report on Carcinogens 1997 Background Document for Saccharin
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`Table 3-1 Summary of Epidemiology Studies Published
`
`Post IARC (1980) •.••..•.••..•••....••..•••..•.•...••••.••••••••..••••••••••25
`
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`4.0 MAMMA.LIA.N CARC:IN"OGENICITY••.•••••••••••••••••••••••••.•••••.••••••••••••••••35
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`4.1 Mammalian Carcinogenicity of Saccharin •.•.•..•.••••••••••••••••••••••••••35
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`4.1.1 Hamsters ......•...•...........•.........•...............•..•..•..........•...••.36
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`4.1.2 Mi~~.•..•••.•••..•••.•.••..•••.•.•••.••••••.•..•••.•••••.•.•••••••••••••••••••••••••36
`
`4.1.3 Rats•.••.•••••.••..••.•.•••..••••..•••.•••.••••.••..••..•••.••.••.••.•••••••••••••••37
`
`4.1.4 Nonhuman Primates ..••••.•••.••••.•.•.•.••.••••••••••.•.•••••••••••••.•••40
`
`Table 4-1 Mammalian Carcinogenicity•••.••••.••.•.••••••••.•.•.••.•••.••.41
`
`4.2 Initiation/Promotion and Co-Carcinogenicity Studies ...•••..•••..•••.•48
`
`4.2.1 Benzo[a]pyrene (BP) •.•.••....••....•.•...••.•...•..........••...•....•....48
`
`4.2.2 N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN) ....•••...••..••48
`
`4.2.3 2-Acetylaminofluorene (AAF) •.....•....••......••....•...•••...••....49
`
`4.2.4 N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide (FANFT) ••••49
`
`4.2.5 N-Methyl-N-nitrosourea (MNU) .•••••••••.•.•••••••.••••••••••••••••••51
`
`4.2.6 Freeze Ulceration ••••••••.•.•••••••••.••••.•••••••••••••••••.••.•••••••••••••52
`
`Table 4-2 Initiation-Promotion and Co-Carcinogenicity
`
`~tll(lies•••••••••••.•••••.•••••••••••.•••.••••••.••.•••.•••••••.•.•••••..••••••••••~~
`
`
`~.() G~~c:)~c:)~CITY••.••••••••••••••.•••••••••••••••••.•••.••••.•••••••••••••••..•..••....••..••••.•6()
`
`~.1 Noneukaryotic Systems •.••.••.••••••••.••.••..••••••••...••••...•••..••..•.•••...•..60
`
`5.1.1 Gene Mutations .••••••••••••••••••••••••••••••••••••••••••••••.••••••••••••••60
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`5.1.2 DNA Damage •.•••.•.•••••.••••••••••••••••••.••••.•••••••••••••••••••••••••.••60
`
`5.1.3 DNA Synthesis .•••••.•.•••.•.•••.•••••••••••••••••.••••••.•••.••••••••••••••••60
`
`5.2 Lower E11karyotic Systems •••••••.•••••.•••••.••••.••••••.•••••••••••••••••••••.••.•60
`
`5.2.1 SaccharoN~Jces cerevisiae ••.•...•.••••••••..••••••.•••••.••••••••••••••••••60
`
`5.2.2 Drosophila melanogaster •••••••••••••••••••••••••••••••••••••••.•••••••••••60
`
`5.2.3 Higher Plants••••.•.•.•••...•.•..••••..•••••••.•••••...••.••.••••••.•.••.••••.•61
`
`5.~ Mammalian Systems In Vitro •••.••.••.••••..••.••••.•••.•••••.•••••••••••••••••..•61
`
`5.3.1 Gene Mutations ••.•.•••.•••••.•••••.••••.••••.•.••••••••••••••••.••••••••••••61
`
`5.~.2 DNA Damage ••••••••••.•••••••••••••••••••.••••••••••••.•••••••..••.••••.•••••61
`
`5.3.3 Inhibition of DNA Repair •.•••••.•.•••••••••.•••.••••••••••••••••••••••••61
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`5.~.4 DNA Synthesis •.•••••.••••••.•••••••••.•••..•••.••••••.•••••••••••.•••••••••••61
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`5.3.5 Chromosomal Damage•••.•.•••.•••.••••••••.••••••••••.••.••••••••••••.••62
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`5.3.6 Cell Transformation.•..••.•...•••••.••....•••••..••••.•.•..•.•••••••••••••.62
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`5.4 Mammalian Systems In Vivo .•••••••••••••••.••••.••••••••••••••••••••••••••••••..•62
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`5.4.1 Gene Mutations and Dominant Lethal Mutations •••••.•••••.62
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`5.4.2 DNA Damage •••••••.•••••.••••••••••••••••••.•••••••••••••••••••••••.•••••••••63
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`5.4.3 Chromosomal Aberrations ..............................................63
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`5.4.4 Induction of Micronuclei .................................................63
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`Table 5-1 Summary of Saccharin Genotoxicity Studies ......••............•.64
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`Figure 5-1 Genetic Activity Profile (GAP) for Saccharin •••.....•••••••••..67
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`Figure 5-2 Genetic Activity Profile (GAP) for Sodium Saccharin .......68
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`Figure 5-3 Schematic View of a Genetic Activity Profile ...........•.......69
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`6.0 OTiffiR RELEVANT DATA ..................................................................70
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`6.1 Absorption, Distribution, and Excretion ......................................70
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`tt.~ M~t=t1Jolism .................................................................................~4
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`6.3 Pharmacokinetics .......................................................................76
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`6.4 Structure-Activity Relationships .........•.•.•••••..........•....••.•••..•....•••76
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`Ci.:i ~ell PJr()lifer-~atiC>n ........................................................................7CJ
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`6.5.1 Hamsters ........................................................................76
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`CJ.~.:l Mi~te................................................................................7Ci
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`CJ.~.~ ~s.ts................................................................................7CJ
`
`ft.~." GuinteJt ~i~s.....................................................................~~
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`6.~.~ NonhumJtn Prims.ttes .......................................................79
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`6.6 ~ell Proliferation with ~a-Administration of Known
`
`~=-r~inogtens ............................................................................80
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`6.6.1 N-Butyl-N-( 4-hydroxybutyl)nitrosamine (BBN) .........•...••.80
`
`6.6.2 2-Acetylaminofluorine (AAF) ....•.....................•...............80
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`6.6.3 N-Methyl-N-nitrosourea (MNU)••••••••••••.••••••••••••••.•••••••••••80
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`Table 6-1 Cell Proliferation ..............................................................81
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`7.() ~~~~ ....................................................................................!)()
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`7.1 Mechanisms of Urinary Bladder Tumorigenesis Found
`
`Predominantly in Male Rats ••••..........••••••........••••.•.....••••••..•...•91
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`7.1.1 The Role of pH in the Promotion of Bladder
`
`Carcinogenesis in Male Rats .......•...........•...........•..........91
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`7.1.2 The Role of Sodium ~oncentration in the Promotion of
`
`Bladder Carcinogenesis in Male Rats ..............................92
`
`7.1.3 The ~ombined Effect of pH Level and Sodium
`~oncentration ................................................................9"
`7.1.4 The Association Between Increased Urinary Output
`
`and Sodium Saccharin-Induced Bladder Tumors ..•........•.94
`
`7.2 Dose Response in ~ell Proliferation and Tumorigenesis ..............95
`
`7.3 Relevance of Animal ~ancers to Humans ...................................97
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`7.3.1 ~omparative Bladder Anatomy and Urine ~hemistry ......97
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`7.3.2 Dose-Response Extrapolation ...•......................................99
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`7.4 Additional Mechanistic Information ..•••.••.........•.•...................... 100
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`7.4.1 Inhibition of Apoptosis .................................................. lOO
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`7 .4.2 Intercellular Communication .................••...•...•...••......•. 1 00
`Table 7-1 Effect of Various Forms of Saccharin on the Rat
`Urinary Bladder •.....•...........•...•.....•....•....••........••.. ~ ••....•....•.... 101
`Table 7-2 Urine Analysis in Rats Given Various Forms of
`Saccharin •••••..•.•.•••..•••••.•••.•.•.•••••..•••.••••.•.••••••••••.••..••••••••.•••••• 1 01
`Table 7-3 Sodium Salts that Produce Urothelial Hyperplasia and
`Increase the Incidence of Bladder Tumors in Rats Fed High
`Doses (> 1 o/o) ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 102
`Table 7-4 Summary of Positive Mammalian Carcinogenicity
`Studies................................................................•...............~.103
`Table 7-5 Interspecies Comparison of the Effects of Sodium
`Saccharin on the Urinary Bladder ...•....••...••..•.•...•••..•••..••...••.. 1 04
`Table 7-6 Interstrain Comparison of the Effects of Sodium
`Saccharin on the Rat Urinary Bladder ................................... 105
`Table 7-7 Interspecies Comparison of Fresh Void
`Urine Chemistry .....•...............•....•.....•....•....•....••........••.•.•...• 1 06
`
`8.0 REFERENCES ....•............................................................................... 107
`
`
`APPENDIX A- Excerpts from the IARC Monograph on the
`Evaluation of the Carcinogenic Risk of Chemicals to Humans
`Volume 22 (Some Non-Nutritive Sweetening Agents),
`Saccharin, pp. 111-185, 1980 ....•.....•.........•.........••...•.....•...•....•...•••.. A-1
`APPENDIX B- Excerpts from the IARC Monograph on the
`Evaluation of the Carcinogenic Risk of Chemicals to Humans
`Supplement 4 (Chemicals, Industrial Processes and
`Industries Associated with Cancer in Humans, IARC
`Monographs Volumes 1 to 29), Saccharin, pp. 224-226, 1982 .......•... B-1
`APPENDIX C- Excerpts from the IARC Monograph on the
`Evaluation of the Carcinogenic Risk of Chemicals to Humans
`Supplement 7 (Overall Evaluations of Carcinogenicity: An
`Updating of IARC Monographs Volumes 1 to 42), Saccharin,
`pp. 334-339, 1987 ••..••....•••..••...•••...•••.••••.••••.••••.•••..••••••••••••.••••••••••••• C-1
`APPENDIX D - Description of Online Searches for Saccharin
`and Saccharin Salts ..•.••..••....••..••••..•...•.••.•••...•.•..•••..•.•..•••.•••.•••••.•••. D-1
`APPENDIX E - Listing of GAP Test Codes in Phylogenetic Order
`for Saccharin and Sodium Saccharin ....•....•...••.•.••...•...••.. E-1
`APPENDIX F- Listing from the Eighth Report on Carcinogens ............•.. F-1
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`NTP Report on Carcinogens 1997 Background Document for Saccharin
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`Proposed Report on Carcinogens Delisting for Saccharin1
`Saccharin is currently listed in the Report on Carcinogens, 8th Edition as reasonably
`anticipated to be a human carcinogen. The basis for this listing was sufficient evidence of
`carcinogeniC-ity in experimental animals. The Calorie Control Council has petitioned the NTP to
`consider delisting saccharin from its Report on Carcinogens based upon mechanistic data related
`to development ofurinary bladder cancers in rats.
`
`Carcinogenicity
`In four studies of up to 30 months duration, sodium saccharin was carcinogenic in Charles
`River CD and Sprague-Dawley male rats as evidenced by a dose-related increased incidence of
`benign or malignant urinary bladder neoplasms at dietary concentrations of 1% or greater (Tisdel
`et al., 1974; Arnold et al., 1980; Taylor et al., 1980; Schoenig et al., 1985). Non-statistically
`significant increases in urinary bladder cancer have also been seen in saccharin-treated female rats
`from studies showing a positive effect in males (Arnold et al., 1980; Taylor et al., 1980).
`Furthermore, several initiation/promotion studies in different rat strains have shown a reduced
`latency and/or increased incidence of similar urinary bladder cancers in male and female rats fed
`sodium saccharin subsequent to treatment with different urinary bladder initiators (e.g., Hicks
`and Chowaniec, 1977; Cohen et al., 1979; Nakanishi et al., 1980b; West et al., 1986; Fukushima
`et al., 1990). Several additional rat studies in which sodium saccharin was administered either in
`the diet or in drinking water were negative for tumorigenicity (Fitzhugh et al., 1951; Lessel, 1971;
`Schmahl, 1973; cited by IARC, 1980; Chowaniec and Hicks, 1979; Hooson et al., 1980; Schmahl
`and Habs, 1984).
`Three mouse studies have reported positive carcinogenicity following exposure to
`saccharin. Two ofthese studies involved surgical implantation of saccharin-containing
`cholesterol pellets into the urinary bladders and resulted in development of malignant urothelial
`neoplasms (Allen et al., 1957; Bryan et al., 1970). In the third study, dietary sodium saccharin
`resulted in increased incidences ofmalignant thyroid neoplasms (Prasad and Rai, 1986). While
`the mouse data cannot be discounted, some ofthese studies had methodological flaws, provided
`limited information, did not show a dose-response, or had unexpected outcomes that may be
`species or strain-specific and should be verified by additional studies. Four studies in mice were
`judged negative for tumorigenesis (Roe et al., 1970; Kroes et al., 1977; Hornberger, 1978;
`Frederick et al., 1989) as were studies in nonhuman primates (McChesney et al., 1977 abstr.;
`Sieber and Adamson, 1978; both cited by IARC, 1980; Thorgiersson et al., 1994; Cohen et al.,
`1996 abstr.) and a single hamster study (Althoff et al., 197 5).
`Much ofthe epidemiology has examined associations between urinary bladder cancer and
`artificial sweeteners, rather than saccharin per se. The time trend data for bladder cancer are
`essentially noninformative with no clear indication that the increased use of saccharin or artificial
`sweeteners commencing in the 1940s is associated with a general increase in bladder cancer when
`controlled for confounding factors, chiefly smoking. Risk ofbladder cancer in diabetics, who
`presumably consume greater amounts of artificial sweeteners compared to the general population,
`
`1Saccharin is produced commercially as calcium and sodium salts as well as the free acid, and the name saccharin
`has been applied to all three.
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`is not greater than risks in the general population (Armstrong and Doll, 1975). Based upon
`several case-control studies there is no overall association between use of artificial sweeteners and
`bladder cancer (reviewed by IARC, 1980; IARC, 1987b; JECF A, 1993). It is harder to reject an
`association between use of artificial sweeteners and bladder cancer in some case-control
`subgroups, even though the numbers are small2 (Howe et al., 1980; Hoover and Strasser, 1980;
`Morrison and Buring, 1980; Cartwright et al., 1981; Morrison et al., 1982; Mommsen et al.,
`1983). Taken together, while the available epidemiology data show no consistent evidence that
`saccharin is associated with increased bladder cancer in general, a small increased risk in some
`subgroups, such as heavy users of artificial sweeteners, cannot be unequivocally excluded. With
`regard to the general population, if sodium saccharin is a risk factor, it is weak and cannot be
`proven or disproved due to lack of actual exposure data and intrinsic limitations of existing
`epidemiology studies.
`
`Other Information Relating to Carcinogenesis or Possible Mechanisms of
`Carcinogenesis
`Extensive studies ofthe mutagenicity and genotoxicity of saccharin have shown generally
`negative but occasionally conflicting results. Sodium saccharin is essentially nonmutagenic in
`conventional bacterial systems but is weakly clastogenic or genotoxic in short-term in vitro and in
`vivo test systems (reviewed by Ashby, 1985; IARC, 1987a,b; Whysner and Williams, 1996)
`with evidence that equimolar ionic solutions of sodium chloride in vitro produce a comparable
`cytotoxic response (Garland et al., 1989a). Urine from mice treated with sodium saccharin was
`mutagenic in the Ames test (Batzinger et al., 1977). Saccharin does not covalently bind to DNA
`and does not induce unscheduled DNA synthesis in bladder urothelium.
`Saccharin-induced carcinogenesis in rats shows a sex predilection for males (Tisdel et al.,
`1974; Arnold et al., 1980; Taylor et al., 1980), an organ specificity for urinary bladder (Tisdel et
`al., 1974; Arnold et al., 1980; Taylor et al., 1980; Fukushima et al., 1983; Schoenig et al., 1985),
`and a dose-response when exposure to dietary concentrations of 1 to 7.5% of the sodium salt of
`saccharin has begun early in life (beginning at birth or immediately at weaning) and is continued
`for approximately two years (Schoenig et al., 1985). The results of mechanistic studies have
`shown that certain physiological conditions must be simultaneously or sequentially present for
`induction of urinary bladder tumorigenesis. These conditions include a urinary pH greater than
`6.5, increased urinary sodium concentration, increased urine volume, decreased urine osmolality,
`presence of urinary crystals or precipitate, and damage to the urothelium resulting in a
`proliferative (hyperplastic) response. All ofthese conditions have been studied extensively in
`male rats but less so in females. The high levels of urinary protein characteristic of many male
`rats may partially explain the sex predilection. The high intrinsic rate of urothelial proliferation
`at about the time of weaning is also believed to contribute to the observed tumorigenic effects.
`The urinary milieu in rats, especially male rats, is sufficiently different from that in humans or
`other species to support the contention that these observations are rat-specific. Pharmacokinetic
`
`2 Morrison and Suring (1980) indicate an increased risk for women. Hoover and Strasser (1980) suggest increased
`risk among low risk (non-smoking, non-occupationally exposed women) and high risk (male heavy smokers)
`subgroups.
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`and metabolism data on sodium saccharin do not explain the male rat sensitivity for induction of
`urinary bladder neoplasms (Sweatman and Renwick, 1979, 1980).
`
`Conclusion
`There is evidence of the carcinogenicity of saccharin in rats but less convincing evidence in
`mice. Mechanistic studies indicate that the observed urinary bladder cancers in rat studies are
`related to urinary pH, osmolality, volume, presence of precipitate, and urothelial damage with
`attendant hyperplasia following dietary concentrations of3% or higher with inconsistent findings
`at lower dietary concentrations. The factors thought to contribute to tumor induction by sodium
`saccharin in rats would not be expected to occur in humans. The mouse data are inconsistent and
`require verification by additional studies. Results of several epidemiology studies indicate no
`clear association between saccharin consumption and urinary bladder cancer. Although it is
`impossible to absolutely conclude that it poses no threat to human health, sodium saccharin is
`not reasonably anticipated to be a human carcinogen under conditions of general usage as an
`artificial sweetener.
`
`Summary References
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`Listing Criteria from the Report on Carcinogens, Eighth Edition
`
`Known To Be A Human Carcinogen:
`There is sufficient evidence of carcinogenicity from studies in humans which indicates a
`causal relationship between exposure to the agent, substance or mixture and human cancer.
`
`Reasonably Anticipated To Be A Human Carcinogen:
`There is limited evidence of carcinogenicity from studies in humans, which indicates that
`causal interpretation is credible, but that alternative explanations, such as chance, bias or
`confounding factors, could not adequately be excluded; or
`
`There is sufficient evidence of carcinogenicity from studies in experimental· animals which
`indicates there is an increased incidence of malignant and/or a combination of malignant and
`benign tumors: (1) in multiple species or at multiple tissue sites, or (2)by multiple routes
`of exposure, or (3) to an unusual degree with regard to incidence, site or type of tumor, or
`age at onset; or
`
`There is less than sufficient evidence of carcinogenicity in humans or laboratory animals;
`however, the agent, substance or mixture belongs to a well-defined, structurally related
`class of substances whose members are listed in previous Reports on Carcinogens as either
`known to be a human carcinogen or reasonably anticipated to be a human carcinogen, or
`there is convincing relevant information that the agent acts through mechanisms indicating it
`would likely cause cancer in humans.
`
`Conclusions regarding carcinogenicity in humans or experimental animals are based on scientific
`judgment, with consideration given to all relevant information. Relevant information includes, but
`is not