SACCHARIN AND ITS SALTS
First draft prepared by
Ms E. Vavasour
Toxicological Evaluation Division
Bureau of Chemical Safety, Food Directorate
Health and Welfare Canada
Ottawa, Ontario, Canada
Saccharin was evaluated by the Committee at its eleventh,
eighteenth, twenty-first, twenty-fourth, twenty-sixth and
twenty-eighth meetings (Annex 1, references 14, 35, 44, 53, 59, and
66). At the twenty-first meeting, the Committee changed the
previously unconditional ADI of 5 mg/kg bw to a temporary ADI of
0-2.5 mg/kg bw and withdrew the conditional ADI of 0-15 mg/kg bw for
dietetic purposes only. This decision was based primarily on
results of animal studies which indicated that excessive and long-
term ingestion of saccharin might represent a carcinogenic hazard.
At the twenty-fourth and twenty-sixth meetings, the temporary ADI of
0-2.5 mg/kg bw was extended pending the completion of ongoing
investigations, including a long-term feeding study in rats and a
large-scale epidemiological study. At the twenty-eighth meeting,
the results of a 2-generation feeding study in rats and
epidemiological data were reviewed and the temporary ADI was again
extended, pending the evaluation of further data on bladder
histopathology from the 2-generation study and information to
elucidate the mechanism by which the compound produced bladder
tumours. These data, along with recent epidemiological studies,
were reviewed at the present meeting, and are summarized in this
2 BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
The disposition of saccharin has been discussed in the previous
monograph and monograph addendum. In addition, a good review of
these aspects of saccharin is presented in Renwick (1985). The main
features of saccharin disposition are presented here.
The disposition of saccharin is influenced by its acidic
properties. With a pKa of 2.2, saccharin exists predominantly in
the un-ionized form in acidic media from which it is more readily
absorbed. It is nearly completely ionized at physiological pH (in
body fluids). Saccharin is more completely absorbed from the
stomachs of species with low pH (guinea pig - pH 1.4; rabbit - pH
1.9) than from those with a higher pH (rat - pH 4.2) (Ball 1973;
Minegishi et al. 1972). In the higher pH of the intestines, it is
slowly absorbed, and rapidly eliminated in the urine. Following
administration of a single oral dose of saccharin in rats and
humans, peak plasma levels of saccharin were rapidly achieved
(Sweatman and Renwick 1980; Sweatman et al. 1981). However,
clearance of saccharin from the plasma was prolonged. Intravenous
administration results in rapid elimination of saccharin in the rat
and human. Consequently, prolonged plasma clearance following oral
administration was attributed to slow and incomplete absorption from
the intestines. The presence of food in the gut was associated with
a reduced initial peak plasma concentration in animals (Matthews
et al. 1973; Sweatman and Renwick 1980) and in man (Sweatman
et al. 1981).
The extent of faecal excretion has been used as an indicator of
unabsorbed saccharin following an oral dose. On the basis of
results from studies of i.v. administration, only a very small
percentage of absorbed saccharin appears in the faeces. Measurement
of the extent of faecal excretion of orally administered saccharin
indicated that gastrointestinal absorption was incomplete and
variable in the rat, with the percentage of the administered dose
recovered in the faeces ranging from 3-39% (Renwick 1985). For the
most part, higher doses were associated with higher faecal
concentrations of saccharin. In humans, 1-8% was recovered in the
faeces following doses of 2 g/person (Sweatman et al. 1981).
Urinary excretion has also been used as a measure of
gastrointestinal absorption since it is the main route of excretion
for absorbed saccharin and since saccharin does not undergo
detectable biotransformation. In rat feeding studies in which
saccharin and its salts were incorporated into commercial rat chow
at levels of 5 or 7.5%, approximately equal amounts of ingested
saccharin were excreted in the urine and faeces (Anderson et al.
1987b; Fisher et al. 1989). By contrast, the administration of
the same level of sodium saccharin in a semi-purified diet (AIN-76A)
resulted in the urinary excretion of 10-20 times more saccharin
compared with the faeces, indicating that absorption from the GI
tract had been more extensive (Fisher et al. 1989).
The gastrointestinal absorption of orally-administered
saccharin in man was 85% based on urinary excretion and area under
plasma concentration-time curves (Sweatman et al. 1981). Almost
80% of the daily dose was recovered in the urine of human volunteers
receiving 1 g of saccharin (as the sodium salt)/day for 4 weeks
(Roberts and Renwick 1985).
Saccharin was found to bind reversibly to plasma proteins
(Renwick 1985). The extent of binding showed a wide range: 3%,
24-35% and 69-86% in the rat and 70-80% in man. Following a single
oral dose to adult rats, saccharin was found to be distributed to
most organs with the highest concentrations in the organs of
elimination (kidney and bladder) followed by the plasma (Matthews
et al. 1973; Lethco and Wallace 1975; Ball et al. 1977;
Sweatman and Renwick 1980). The steady-state concentrations of
saccharin in adult male rats fed 1-10% saccharin in the diet were
consistent with the observations from these single-dose studies.
There is no evidence of bioaccumulation of saccharin in any tissue.
Placental transfer of saccharin to the fetus has been observed in
rats (Ball et al. 1977), monkeys (Pitkin et al. 1971) and humans
(Cohen-Addad et al. 1986).
As indicated above, the urine is the principal route of
elimination for saccharin after both oral and parenteral dosing.
Renal tubular secretion is the major mechanism of elimination in
both rats and humans as indicated by the reduction in plasma
clearance of saccharin when administered with probenecid, an
inhibitor of the renal tubular secretion of anions. Glomerular
filtration is not considered to be as important a mechanism due to
the high degree of plasma protein binding of saccharin. Renal
tubular secretion is a saturable process and plasma concentrations
of saccharin greater than 200 µg/ml have been associated with
saturation in the rat (Sweatman and Renwick 1980). Dietary levels
of saccharin exceeding 5% resulted in accumulation of saccharin in
the plasma and tissues due to decreased renal clearance. However,
decreased renal clearance was not detected following administration
of an oral dose of 2 g in humans which produced a peak plasma
concentration of 40 µg/ml (Sweatman et al. 1981).
The consensus of the most recently conducted research in a
number of experimental species or humans is that saccharin is not
metabolized (Renwick 1985). In addition, radiolabelled saccharin
did not bind to the DNA of the liver or bladder of rats in vivo
(Lutz and Schlatter 1977), indicating that saccharin was not
metabolized to an electrophilic compound.
2.1.3 Effects on enzymes and other biochemical parameters
The high concentrations of sodium saccharin in the lumen of the
gastrointestinal tract due to dietary administration of sodium
saccharin resulted in the decreased activity of a number of
digestive enzymes of the pancreas and intestines. Saccharin is an
inhibitor of urease and proteases in vitro (Lok et al. 1982) and
feeding saccharin in the diet causes accumulation of protein and
tryptophan and its metabolites in the caecum (Sims and Renwick
Saccharin also led to the inhibition of carbohydrate digestion
which resulted in the faecal elimination of polysaccharides. The
in vitro activities of amylase, sucrase and isomaltase were
inhibited by the presence of saccharin (Renwick 1989).
Feeding male rats a diet containing 5% sodium saccharin for 14
days did not result in induction of hepatic cytochrome P-450
(Hasegawa et al. 1984).
The administration of 7.5% sodium saccharin to rats in both
1- and 2-generation feeding studies had no effect on hepatic
concentrations of cytochrome P-450, cytochrome b5, cytochrome P-450
reductase, arylhydrocarbon hydroxylase activity or glutathione
content per mg protein. Hepatic dimethylnitrosamine-N-demethylase
activity was increased in both neonatal and adult male and female
rats fed a high dietary concentration of sodium saccharin (Heaton
and Renwick 1991a).
Sulfate conjugation of phenol in vivo was reduced in both
male and female rats fed a 7.5% sodium saccharin diet in a
2-generation protocol. The maximum effect was detected in neonatal
animals at 5 weeks of age. Dietary supplementation with cysteine
restored sulfate conjugation, indicating that the effect was caused
by poor availability of sulfur-containing amino acids rather than
inhibition of the sulfotransferase enzyme (Heaton and Renwick
2.2 Toxicological Studies
2.2.1 Long-term toxicity/carcinogenicity studies
There have been no additional 2-generation carcinogenicity
studies on saccharin since the IRDC study which was available as
unpublished data at the twenty-eighth meeting in 1984. The data for
this study have been published (Schoenig et al. 1985). The tumour
incidence data for the urinary bladder of F1 male rats were the
same as those reported in the previous monograph addendum (Annex 1,
reference 67) with the exception that the incidence of total tumours
at 3% sodium saccharin in the diet was 1.7%, not 1.6% as then
reported. The no-observed effect level was 1% in the diet, although
the application of a threshold dose-response model to the data
suggested a threshold close to 3% (Carlborg 1985).
The bladder histology studies have been re-evaluated under
blind conditions with the result that higher incidences of
papillomas and carcinomas were reported in both the control and
lower treatment groups and the apparent increases in transitional
cell carcinomas and combined neoplasms in the 3% group were found to
be not statistically significant (p=0.25 and 0.41, respectively).
The blind reevaluation confirmed that the 1% dietary level of sodium
saccharin had no carcinogenic nor proliferative effect on the
bladder epithelium (Squire 1985).
Although no new 2-generation study has been reported in rats or
other species, studies on promotion of bladder carcinogenicity used
control groups in which the animals received 5% sodium, calcium and
acid saccharin in the diet without treatment with an initiator.
After 2 years or 72 weeks, respectively, of feeding with sodium
saccharin, the incidence of simple hyperplasia was higher than that
in untreated controls. The incidence of papillomas and carcinomas
of the bladder was comparable in the sodium-saccharin-treated groups
and untreated controls (Hasegawa et al. 1985; Cohen et al.
Nodular hyperplasia but no papillomas or carcinomas were
detected in the bladder epithelium of male rats given 5% sodium
saccharin in the diet for a period of 112 weeks, starting at 7 weeks
of age (Hibino et al. 1985).
2.2.2 Special studies on promoting activity
The promotion of known bladder carcinogens by high dietary
concentrations of sodium saccharin has been detected only in rats
(Fukushima et al. 1983a). Administration of 5% sodium saccharin
in the diet does not increase the incidence of bladder tumours in
mice initiated with 2-acetylaminofluorene (Frederick et al. 1989).
High dietary concentrations of sodium saccharin (5.0% of the
diet or 2.0 g/kg bw/dy from drinking water) promoted the effects of
known bladder carcinogens such as methyl-N-nitrosourea (MNU) (Hicks
et al. 1973), N-[4-(5-nitro-2-furyl)-thiazolyl]formamide (FANFT)
(Cohen et al. 1979), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)
(Nakanishi et al. 1980b) and 2-acetylaminofluorene (AAF)
(Nakanishi et al. 1982). Feeding rats with high concentrations of
sodium saccharin following an insult such as freeze ulceration
causes increases in nodular hyperplasia (Murasaki and Cohen 1983)
and in bladder tumours (Hasegawa et al. 1985). Promotion of the
effect of bladder carcinogens is not sex-specific and has been
reported in both male (Cohen et al. 1979) and female (Hicks et
al. 1978) rats. The sex specificity of promoting activity has
been studied in male and female F344 rats given BBN as an initiator
and up to 5% sodium saccharin as the promoter. Papillomas and
carcinomas were not detected, but both sexes showed a similar dose-
response for sodium saccharin-related simple hyperplasia and
papillary or nodular hyperplasia (Nakanishi et al. 1980a).
Administration of a diet containing 5% sodium saccharin to rats
increased DNA synthesis in the urinary bladder, but not in liver or
forestomach. It did not affect ornithine decarboxylase activity (a
molecular marker of tumour promotion) in these organs (Tatematsu
et al. 1986). An increase in DNA synthesis in the urothelium is
produced by a number of promoters of diverse structure (Shibata
et al. 1989c).
Transitional cell carcinomas of the urinary bladder in rats
initiated with FANFT treatment for 4 or 6 weeks followed by long-
term treatment with sodium saccharin or other promoters have been
analyzed for the presence of H-ras gene activation. There was
evidence of increased expression of the ras gene product, p21, in
transitional cell carcinomas and of H- ras gene mutations. The
compounds used in the promoting phase had essentially no effect on
H- ras mutation, and the authors concluded that the effects
observed were due to the FANFT initiation phase (Masui et al.
Proto-oncogenes may contribute to the development of malignancy
when their structure or expression is altered, and ras gene
activation in particular has been demonstrated in 5-17% of human
urinary tract tumours (Fujita et al. 1984, 1985). Similar
activation of the ras gene has been demonstrated in animals
treated with the bladder carcinogens BBN and FANFT (Jones and Wang
1989; Sawczuk et al. 1987). The expression of ras p21 has been
studied using immunohistochemical techniques in normal urothelium as
well as in urothelial lesions in rats from long-term promotion
studies using FANFT. Immunoreactivity with ras p21 antibody was
demonstrated in urinary bladder lesions of more than 50% of rats
treated with FANFT alone or FANFT followed by sodium saccharin
(0.2 or 5.0% in the diet). Immunoreactivity to ras p21 was not
observed in rats treated with sodium saccharin alone. More recent
studies by the same authors have confirmed the mutational activity
of the H- ras gene in rat urinary bladders induced by FANFT and the
lack of any effect of sodium ascorbate or sodium saccharin on this
activation (Masui et al. 1990, 1991).
2.2.3 Special studies on genotoxicity
The results of genotoxicity studies with saccharin are summarized in Table 1.
Table 1. Genotoxicity studies with saccharin.
Test System Test Object Concentration Results Reference
Cell mutation/ouabain Human RSa cells 10 - 22.5 mg/ml Positive Suzuki and Suzuki 1988
In vitro chromosomal Chinese hamster 8 - 16 mg/ml Positive Ashby and Ishidate 1986
aberration lung fibroblasts
In vivo chromosomal ICR/Swiss male mice 0, 0.5, 1.0 and
aberration 1.5 g/kg bw/day, Positive Prasad and Rai 1987
p.o. for 24 weeks
Dominant lethal ICR/Swiss male and 0, 1 and 2 g/kg Positive Prasad and Rai 1986
female mice bw/12 h x 5, p.o.
Insect genotoxicity Drosophila melanogaster, 0.5, 5.0 and 50 mg Negative Lamm et al. 1989
meiosis repair deficient in nutrient media
A review by Ashby (1985) presents evidence that the positive
results obtained in genotoxicity studies with saccharin, mostly
showing clastogenicity, probably do not involve covalent interaction
of saccharin with nuclear DNA, but are more probably the result of
ionic imbalances at the high concentrations used in the assays. The
low systemic toxicity of saccharin to mammals and their constituent
cells in culture has allowed the use of exceptionally high dose
levels in genotoxicity assays. It was concluded that the structural
disturbances of eukaryotic cells in vitro and very weak
intermittent activity in vivo were equivalent to and comparable to
the genotoxic profile for sodium chloride.
The different salts of saccharin (at 8-16 mg/ml) showed equal
clastogenic activity in Chinese hamster lung cells (Ashby and
Ishidate 1986), indicating that both ionic and osmotic changes to
the medium may be critical determinants of the observed clastogenic
effects. Mutagenicity and/or chromosome aberrations due to sodium
chloride at high concentrations have been demonstrated recently
using mouse lymphoma cells (Brusick 1986; Moore and Brock 1988) and
Saccharomyces cerevisae (Parker and von Borstel 1987). Sodium
chloride was included as a control in a study on human RSa cells and
gave a small non-significant increase at a concentration equivalent
to the high concentration of sodium saccharin (0.11M) (Suzuki &
Suzuki 1988). However, the results of this study are difficult to
interpret because the prolonged exposure to high concentration of
sodium ion and chloride ion may have affected Na+/K+-ATPase
activity, and sodium and chloride channels, thereby altering
intracellular sodium concentration and sensitivity to ouabain.
Positive results have also been reported in 2 recent studies in
which mice were dosed with solutions prepared by dissolving
commercial saccharin tablets in water (no information was given on
the salt form, purity or the excipients present). In one study, a
dose-related increase in the incidence of chromosomal abnormalities
in bone marrow and meiotic cells (not specified) was reported
(Prasad and Rai 1987). This finding contrasts with a report that
administration of saccharin at 20 g/L (salt not specified) in
drinking water for 100 days affected neither bone marrow cells nor
dividing spermatocytes (Leonard and Leonard 1979).
The second study using commercial saccharin tablets involved a
dominant lethal test. An increased incidence of dead implants was
reported in the females mated with saccharin-treated male mice
(Prasad and Rai 1986).
A positive dominant lethal test had been reported previously in
a study in which mice were treated with a 1.72% solution of sodium
saccharin, again prepared by the dissolution of commercial saccharin
tablets manufactured in India and administered as drinking water
(Sanjeeva Rao and Qureshi 1972).
In contrast to these studies, pure sodium saccharin has been
shown to be negative in dominant lethal studies using male mice
treated at 5 g/kg/day for 5 days (Machemer and Lorke 1973); using
male and female mice treated at 2 g/kg/day for 10 weeks (Lorke and
Machemer 1975); and using male mice given either a single
intraperitoneal injection (2 g/kg) or 2% saccharin in the drinking
water for 100 days (Leonard and Leonard 1979). The positive
findings also conflict with the negative data from multi-generation
feeding studies in mice (Kroes et al. 1977).
2.2.4 Special studies on cell transformation
Malignant transformation of cultured human foreskin fibroblasts
occurred when the cells were exposed to a non-toxic concentration of
sodium saccharin (50 µg/ml) after being released from the G1
phase, followed by exposure to either N-ethyl or
N-methylnitrosourea. The combination of nitrosourea and saccharin
was necessary for the observation of transformation (Milo et al.
Prolonged exposure (up to 89 days) to sodium saccharin (6 or
12 mM; approximately 1-2 mg/ml) caused hyperplastic and other
abnormal cellular changes in cultures of bladder epithelial cells
from female rats (Knowles et al. 1986). A subsequent study
(Knowles and Jani 1986) showed that treatment of the culture with
N-methylnitrosourea (MNU) resulted in the appearance of
preneoplastic epithelial foci and that treatment with sodium
saccharin (12 mM) following a low dose of MNU (25 µg/ml) resulted in
a very small but apparently significant significant increase in such
foci. The incidences of such lesions were 0/896 for MNU alone and
3/1096 for MNU followed by saccharin. The parameters for
determining significance were not indicated. No concurrent controls
were run for this experiment and the incidence in controls from a
different experiment run in the same series was 6/863. In contrast
to the foci produced by high doses of MNU alone (250 µg/ml), none of
the foci produced by MNU (25 µg/ml) followed by saccharin gave cell
lines that were tumorigenic in vivo.
The combined effects of sodium saccharin with MNU on in vitro
explants of female rat bladders were also studied. MNU alone
exhibited severe and extensive cytotoxicity to both the urothelium
and stroma, while sodium saccharin (0.5% in the medium) as well as
sodium cyclamate and cyclophosphamide produced changes in the
urothelium consistent only with hyperplasia, demonstrating globular
and pleomorphic microvilli. Treatment with a low dose of MNU (100
µg/ml) after exposure to sodium saccharin (0.1 or 0.5% for 28 days)
elicited more extensive abnormalities. On the basis of these
in vitro data, the authors suggested that saccharin may have
initiating activity in a multistage process, a conclusion which is
at variance with the large body of information on the mechanism of
action of saccharin (Norman et al., 1987).
Sodium saccharin (6 mM) did not produce proliferating
epithelial foci in bladder explants from female rats but increased
the numbers of foci in explants treated with MNU (50 or 100 µg/ml)
prior to exposure to saccharin. Cultured cell lines from foci
derived from explants treated with MNU alone and MNU + saccharin
formed tumours when injected into mice (Nicholson and Jani 1988).
The urothelial transforming activity of sodium saccharin
in vitro has been studied using epithelial cells derived from male
rat bladders and treated with 2-amino-4-(5-nitro-furyl)thiazole
(ANFT), a water-soluble metabolite of FANFT. Prolonged treatment
with ANFT (1 µg/ml), but not sodium saccharin (25 µg/ml),
transformed the cells in vitro as evidenced by morphological
changes, the ability to grow on plastic, and tumorigenicity when
injected into mice. Exposure to sodium saccharin and ANFT produced
effects similar to ANFT alone, while urea (0.05%) may have enhanced
the effects of ANFT. The absence of "promotion" in this study
compared with those presented above may have arisen from the lower
concentration of sodium saccharin employed (Mann et al. 1991).
A model of promotion of bladder carcinogenicity has been
reported in which male Fischer 344 rats were given the bladder
carcinogen BBN (0.05%) for 3 weeks, followed by the possible
promoter for 9 weeks. At termination, the urinary bladders were
removed, digested with collagenase and DNAase and the number of
colonies able to grow on double soft agar was determined. Growth in
double soft agar is considered indicative of transformation
(Hamburger and Salmon 1977; Colburn et al. 1978). In this test
system, 9 weeks of dietary administration of 5% sodium saccharin, 1%
D-tryptophan, DL-, D-, or L-leucine and DL- or L-isoleucine all
significantly increased the numbers of colonies growing in vitro.
Dietary administration of 2% L-tryptophan did not significantly
increase the numbers of colonies. Treatment of the rats with BBN
for 6 weeks in the absence of a putative promoter resulted in a high
yield of colonies (Hashimura et al 1987).
High concentrations of the sodium, potassium and calcium salts
of saccharin (100-200 mM; equivalent to 18-36 mg/ml) are toxic to
the AY27 line of transformed rat bladder epithelial cells.
Comparable toxicity was shown by sodium ascorbate but not by sodium,
potassium or calcium chlorides. The authors claim that these
observations are of relevance because such high concentrations of
sodium and saccharin are present in the urine of rats fed saccharin-
containing diets. However, the intra-cellular concentration of the
saccharin anion in vivo is much closer to that of plasma which is
about 10-fold less than that in urine (Garland et al. 1989a).
2.2.5 Special studies on the effect of saccharin on urine
composition and bladder epithelial proliferation
Statistically significant, dose-related changes in urinary
parameters (increased Na+ concentration, increased urine volume
and decreased osmolality) have been found to precede and to occur in
association with tumour development in the urinary bladder of male
rats. A large number of studies have been conducted to establish
the effect of factors modifying these non-neoplastic responses on
subsequent outcomes of the treatment on the bladder epithelium.
184.108.40.206 Salt form
Feeding different salts of saccharin (sodium, potassium,
calcium and acid) for 10 weeks resulted in different effects on
[3H]-thymidine labelling in the urinary bladder epithelium
(Hasegawa & Cohen 1986). Dietary ingestion of the sodium salt
resulted in the highest labelling index (0.6±0.2%), the potassium
salt resulted in a weak, but significant, effect (0.2±0.1%), the
calcium salt resulted in a questionable and non-significant effect
(0.1±0.1%), while the acid form (0.07±0.04%) gave a result similar
to controls (0.06±0.04%). In each of these cases, the bladder
epithelium was exposed to similar concentrations of the saccharin
anion in the urine. Dietary ingestion of the sodium and potassium
salts of saccharin were associated with an increased urine volume
and slightly higher urine pH (sodium salt only) compared with
untreated controls, while a decrease in urine pH and no change in
urine volume was found in the calcium and acid saccharin groups.
These findings were essentially confirmed in a subsequent study
in which male rats were fed diets containing 200 µmol/g of the
different salt forms (equivalent to 5% sodium saccharin) for 10
weeks. Simple hyperplasia of the bladder epithelium was noted in
the rats ingesting the sodium and potassium salts, but not in those
ingesting the calcium or free acid forms. The effect was independent
of the total urinary saccharin or urinary concentration of saccharin
(Anderson et al. 1988a).
Based on the finding that the sodium, potassium, calcium and
acid forms of saccharin differed in the extent to which they
produced epithelial proliferation in the bladder of the male rat in
the presence of similar concentrations of saccharinate anion,
Williamson and coworkers investigated the possibility that
differences in the ionic concentration of urine could result in
differences in the electronic structure of the saccharin molecule
itself. Using nuclear magnetic resonance spectroscopy, the
electronic structure of the saccharin molecule was observed in the
presence of varying concentrations of hydrogen, potassium, sodium,
calcium, magnesium, bicarbonate and urate ions. The presence of
these ions at physiological levels did not significantly alter the
electronic structure of the saccharin molecule (Williamson et al.,
A recent study has extended these short-term observations to a
full initiation-promotion study in which male rats were given 0.2%
dietary FANFT for 6 weeks followed by various treatments for 72
weeks. Treatments included doses of sodium saccharin equivalent to
the bottom (3%) and middle (5%) of the dose-response as reported in
the IRDC 2-generation bioassay (Schoenig et al. 1985). Sodium
saccharin in Prolab diet produced a dose-related increase in the
incidence of bladder carcinoma over that in FANFT-initiated
controls. Calcium saccharin also produced a statistically
significant increase of bladder carcinomas but equimolar dietary
concentrations of acid saccharin did not produce a significant
increase in the incidence of bladder tumours.
The observation that co-administration of ammonium chloride
(NH4Cl) with 5% sodium saccharin abolished the promoting activity
was of equal or greater significance to the data for the different
salt forms of saccharin. The urine pH of animals given sodium
saccharin following FANFT initiation was slightly, but significantly
higher (by about 0.1 - 0.2 pH units) than that of the corresponding
controls, while calcium saccharin caused a slight decrease in urine
pH. In contrast, the urine pH of animals given saccharin acid or
sodium saccharin + NH4Cl was 1 pH unit less than that of the
corresponding controls. The study also showed that administration
of calcium saccharin with sodium chloride or sodium saccharin with
calcium carbonate resulted in promoting activity similar to that
with sodium saccharin alone. In addition, sodium chloride itself
had a significant tumour-promoting effect. The authors concluded
that the enhancing factors for promotion of bladder carcinogenesis
in the rat by compounds such as sodium saccharin are a high urinary
sodium concentration, a high urinary pH (>6.5) and possibly an
increase in urine volume (Cohen et al. 1991).
A study in which a range of compounds was given to male F344
rats for 16 weeks reported that 5% sodium saccharin caused an
approximately 5-fold increase in labelling index. Sodium
bicarbonate produced a 10-fold increase and a combination of sodium
saccharin and sodium bicarbonate gave an approximately additive
effect (Debiec-Rychter and Wang 1990).
220.127.116.11 Anion specificity
High dietary concentrations of the sodium salts of other
organic acids (>1%) have also been tested for their ability to
enhance DNA synthesis or hyperplasia of the urinary epithelium of
the rat or act as promoters of bladder carcinogenesis.
In initiation-promotion models, the sodium salts of the
following compounds were found to act as promoters of bladder
carcinogenesis in the rat: ascorbate (Fukushima et al. 1983b,
1983c, 1984, 1986a and b); Cohen et al. 1991b); erythorbate
(Fukushima et al. 1984); o-phenylphenate (Fukushima et al.
1983d); citrate (Fukushima et al. 1986c); bicarbonate (Fukushima
et al. 1986a, 1988a). All of these salts increased urine pH and
sodium excretion compared with untreated controls. Promotion of
bladder carcinogenesis was not caused by the corresponding acids
erythorbic acid (Fukushima et al. 1987a); o-phenylphenol
(Fukushima et al. 1983d); or nitrilotriacetic acid (Kitahori
et al. 1988).
Sodium hippurate, which was not a promoter, increased urinary
sodium excretion, but did not significantly increase urine pH
(Fukushima et al. 1983b, 1986b).
One study did not show a promoting effect for sodium citrate or
sodium ascorbate in an initiation-promotion protocol despite the
expected increase in urinary sodium concentrations and demonstrated
increase in urine pH (Inoue et al. 1988). The period for
promotion in this study was 20 weeks as compared with 32 weeks in
the positive studies.
Co-adminstration of ascorbic acid with salts which increased
urine pH and elevated urinary sodium concentrations (sodium
bicarbonate or potassium bicarbonate but not calcium carbonate or
magnesium carbonate), resulted in promoting activity (Fukushima
et al. 1986a, 1987b, 1988a, 1988b). Administration of the sodium
salts of ascorbic acid and nitrilotriacetic acid in conjunction with
ammonium chloride (sodium ascorbate and trisodium nitrilotriacetate)
resulted in decreased urinary pH and decreased promoting activity
(Fukushima et al. 1986a; Kitahori et al. 1988).
These data showing the importance of urine pH are consistent
with the report that co-administration of sodium ascorbate with
sodium saccharin enhances bladder tumour promotion in male rats.
The co-administration of ascorbic acid with sodium saccharin both
lowered the urine pH (by about 1 pH unit) compared with sodium
saccharin on its own and abolished the promoting activity (Fukushima
et al. 1990)]
Similar results were obtained in studies with administration of
high doses of organic acids either alone or in combination with
other acidifying and alkalinizing salts using increased DNA
synthesis and urothelial hyperplasia as the endpoints. Those
treatments which caused a marked increase in urinary pH, sodium
concentration and urine volume were associated with increased
epithelial hyperplasia of the urinary bladder (5% ascorbic acid -
Shibata et al. 1989b; 6% monosodium glutamate - De Groot et al.
Sodium bicarbonate fed at a level of 0.64% of the diet for 104
weeks study resulted in elevated urinary pH and Na+ concentration,
but did not result in pleomorphic microvilli or a significant
increase in bladder tumour incidence (Fukushima et al. 1989).
These findings suggest that although elevated urinary sodium
concentrations and elevated pH are necessary co-factors in bladder
tumour promotion, they are not sufficient stimulus for bladder
tumour formation in the absence of an initiator.
18.104.22.168 Urine volume
The possible promoting activity arising from an increase in
urine volume without a marked change in urine pH or sodium ion
concentration has been studied using the diuretics acetazolamide and
furosemide. Acetazolamide (0.35%) decreased the urine osmolality
and increased fluid intake but lacked promoting activity in one
study (Fukushima et al. 1983b). However, that study showed
limited sensitivity in demonstrating the effects of 5% sodium
saccharin and 5% sodium ascorbate.
A more recent initiation-promotion study using acetazolamide
(0.35%) has reported significant promoting activity, but these
effects were not shown to be independent of a change in pH since
acetazolamide also increased the urine pH from 6.7 to 7.4 (Masui
et al. 1988b).
Furosemide treatment 250 mg/kg, 3 times weekly for 32 weeks)
was not a promoter of bladder carcinogenesis in male F344 rats. The
dose produced an increase in urine volume, a slight increase in
urine pH, but did not increase urinary sodium ion concentration
(Shibata et al. 1989a). These data suggest that an increase in
urine volume in the absence of changes in pH and sodium ion
concentration does not result in the promotion of bladder
22.214.171.124 Diet and rat strain
Feeding rats 5 or 7.5% sodium saccharin in commercial diets,
i.e. Prolab and Purina for 4 or 10 weeks resulted in an increased
[3H]thymidine labelling index in the bladder epithelium. However,
a greatly diminished or negligible increase in labelling index was
observed when the same concentrations were incorporated into NIH-07
(a crude cereal-based diet) or AIN-76A (a purified diet) diets.
Diet-related differences in urine volumes and urine pH were
implicated as being contributory factors since rats fed 7.5% sodium
saccharin in the AIN-76A diet had the lowest urine volume and the
lowest pH compared with Purina diet. The urinary concentration of
saccharin per se was highest in the group fed saccharin in the
AIN-76A diet (this was the group which showed no increase in
labelling index) indicating that the urinary concentration of
saccharin is not a critical factor. The F344 strain was more
sensitive to saccharin-induced hyperplasia and increases in the
[3H]-thymidine labelling index than were Sprague-Dawley rats
(Garland et al. 1989b).
In a similar study, sodium and calcium saccharin were fed to
male F344 rats at 5% in either Prolab 3200 or AIN-76A diets to
assess the effects on urinary parameters. The urine volume was
actually greater in rats fed the AIN-76A diet containing saccharin
than in the corresponding Prolab group and the urinary saccharin
concentration was less. The urine pH was about 1.5 units lower in
the urine from rats fed with the AIN-76A diets compared with those
fed the Prolab diets (Fisher et al. 1989).
A full initiation-promotion study with sodium saccharin has
shown that dietary levels of 5% do not act as a promoter of bladder
carcinogenesis if given to rats in the AIN-76A diet (Okamura et al.
1991). This is consistent with an earlier observation with this
diet (Imaida and Wang 1986).
The basal diet has been shown to play a similar critical role
in the promotion of bladder carcinogenesis by sodium ascorbate (Mori
et al. 1987). The nature of the basal diet also influences the
urinary changes associated with the administration of monosodium
glutamate (De Groot et al. 1988).
2.2.6 Special studies on the basis of sex/species specificity of
carcinogenic effects of saccharin
Crystals and flocculent precipitate have been observed in the
urine of rats fed sodium saccharin for four weeks (Cohen et al.
1989). Milky flocculent precipitate has also been noted in the
urine of rats fed 5% sodium saccharin in a chronic study which was
more pronounced in males than in females and was found to contain
saccharin and protein (Arnold et al. 1980).
In preliminary evaluations, a correlation was demonstrated for
individual rats between early appearance of the precipitate,
consistent appearance over the course of the study and the
subsequent severity of "bladder effects" over a 10-week treatment
period (cited Cohen and Garland 1992).
A number of methods have been used to quantify this urinary
precipitate. Visual inspection of the filtered urine showed that a
white precipitate was frequently noted on the filters from male rats
treated with sodium saccharin, but rarely on the filters from
control male rats, control and treated female rats and control and
treated male and female mice. In addition, precipitate was not noted
in the filtered urine of rats treated with acid saccharin. A
turbidity assay which measured absorbance of the urine at 620 nm
showed that urine turbidity was increased by feeding of sodium
saccharin to rats (information on sex not available but presumed to
be male), at levels exceeding 3% and with sodium ascorbate at 6.84%.
The urine turbidity of rats treated with lower levels of saccharin
or with acid saccharin, ascorbic acid, sodium saccharin with
ammonium chloride or sodium saccharin in AIN-76A semi-synthetic diet
was similar to that in untreated rats (cited in Cohen and Garland
Gel filtration of the protein component of the urinary
precipitate showed it to migrate with low molecular weight proteins
including alpha2u-globulin (Cohen et al. 1990). Binding of
saccharin to urinary proteins is minimal at a pH of 5.5, but
increases with increasing pH. Treatments which were associated with
a urinary pH less than 6.5 did not result in the formation of a
urinary precipitate or in the production of proliferative changes in
the bladder (cited in Cohen and Garland 1992).
A study was conducted to determine whether alpha2u-globulin
plays a role in the development of bladder lesions in sodium
saccharin-treated rats. The effects of sodium saccharin on urinary
parameters and bladder morphology were compared in NCI-Black-Reiter
(NBR) male rats which do not synthesize alpha2u-globulin, castrated
male F344 rats which have lower alpha2u-globulin levels, and intact
male F344 rats. Scanning electron microscopy and light microscopy
showed that 7.5% sodium saccharin in the diet had less of an effect
on the bladders with respect to morphological changes (simple and
proliferative hyperplasia) of the NBR rats than in the intact F344
rats, and the results from the castrated F344 rats were intermediate
between the intact F344 and NBR male rats. The ability of saccharin
to bind in vitro to urinary proteins was much less with NBR rat
urine than with urine from intact F344 rats. Urine from castrated
F344 rats was not tested. Binding was predominantly to proteins of
low molecular weight in the F344 rat and equally divided between low
and higher MW proteins in the NBR rat. Although urine volume and
sodium concentration were elevated, urine pH was actually decreased
in sodium saccharin-treated rats as compared with respective
controls, but still exceeded values of 6.5. The NBR rats had in
general a much greater urine volume, so that urinary concentration
of solutes was decreased. Visual inspection indicated that a white
precipitate was occasionally present in the urine of untreated rats,
in the majority of urine samples collected from sodium saccharin-
treated intact and castrated F344 rats and in approximately half of
the sodium saccharin-treated NBR rats. The presence of
alpha2u-globulin was detected using Western blot analysis in the
urine from intact male F344 rats, very low levels in the urine from
female F344 rats and none in the urine from NBR rats. Data for the
castrated rats were not available from this study, but levels are
reported to be intermediate between those of intact and NBR rats.
The results of this experiment demonstrated a relationship between
urinary alpha2u-globulin levels, binding of saccharin to urinary
proteins and morphological changes in the bladder (Garland et al.
The Committee was not convinced that this evidence showed that
alpha2u-globulin has a role in bladder carcinogenesis.
2.2.7 Special studies on the possible significance of exposure to
saccharin through lactation
The physiological changes in young male rats that had been
exposed to sodium saccharin from parturition (up to 5% in the diet)
have been compared to those detected in animals exposed only from
weaning and to controls. The results at 10 weeks after weaning
showed that exposure during lactation slightly enhanced the effects
of saccharin compared to animals exposed only from weaning. The
animals exposed from parturition showed a significantly lower urine
osmolality, lower body weight and lower food intake and a non-
significant increase in urine mass, bladder mass and bladder
hyperplasia compared with those exposed after weaning (Anderson
et al. 1988b).
Administration of a diet containing 7.5% sodium saccharin
during a 2-generation protocol resulted in anaemia in both dams and
pups and a severe reduction in post-natal body weight (35% by day
30). This observation had been made previously in weanling rats
from the IRDC long-term study (Schoenig et al. 1985). Saccharin-
treated pups, 28-30 days old, showed the typical saccharin-related
changes in the gastrointestinal tract (increased caecum weight and
moist faeces) and in the urine (increased volume and decreased
osmolality, increased Na+ and decreased K+ and Ca++). Pups
raised on saccharin-containing diets had elevated serum
concentrations of cholesterol, triglycerides and Vitamin E, and
decreased concentrations of Vitamin A and folate in the serum and
liver compared with untreated controls (Garland et al. 1991a).
A subsequent investigation at dietary concentrations of 0, 1, 3
and 7.5% sodium saccharin using a two-generation protocol,
investigated these findings in 30-day-old pups in more detail. The
effects of sodium saccharin on anaemia, serum folate and Vitamin A
were dose-dependent, while the effect on serum concentrations of
vitamin E, cholesterol and triglycerides was biphasic, with a
decrease at 1% and 3% and an increase at 7.5% in the diet. These
effects were mostly reversible by 90 days of age. At 7.5% in the
diet, there was a decrease in liver weight which was associated with
a decrease in glycogen and an increase in the numbers of lipid
vacuoles. The dietary NOEL for sodium saccharin-induced changes in
the liver and for anaemia was estimated to be 1% (Garland et al.
These nutritional and biochemical effects resemble findings in
pups of iron-deficient dams, and further studies were conducted to
see whether iron and/or folate supplementation could counteract the
effects of 7.5% dietary sodium saccharin in pups up to 30 days old.
Iron supplementation reversed some of the biochemical changes, but
had no effect on the majority of the urinary changes which are
typically observed with high dietary levels of sodium saccharin and
implicated with enhancing the epithelial hyperplasia of the bladder.
It is likely that the biochemical changes in the neonatal rat
described above are a consequence of iron deficiency which is an
indirect effect of sodium saccharin treatment and probably
independent of urinary and bladder effects (Garland et al. 1992b).
Cohen and his associates have proposed that during the neonatal
period newborn rats are uniquely sensitive to the mitogenic effects
of saccharin on the urothelium. In this model, the importance of
exposure to sodium saccharin during the this period arises from the
fact that approximately one third of the total lifetime mitoses of
the urothelium occur within the first 3 weeks of life (Cohen and
Ellwein 1991). A significant increase in cell proliferation rates
due to sodium saccharin administration during the 3 weeks after
birth (Masui et al 1988a), coupled with the background probability
of spontaneous genomic errors would substantially increase the
number of initiated cells (Cohen and Ellwein 1990).
The critical event(s) occurring during the neonatal phase,
leading to an increased population of initiated cells, has not been
2.2.8 Special studies on the effects of saccharin on digestion
The caecal enlargement which results from feeding high dietary
concentrations of saccharin to rats is accompanied by an increase in
the total numbers of micro-organisms (Mallett et al. 1985). Since
saccharin has been shown to inhibit the activity of digestive
enzymes mediating hydrolysis of complex carbohydrates, as well as
that of several proteases and urease (Section 2.1.3), this increase
in the numbers of caecal bacteria suggests a large increase in
nutrient availability for microorganisms in the lower GI tract.
A study was performed to investigate a possible connection
between the saccharin-mediated decrease in hydrolysis of complex
carbohydrates and altered urinary parameters (increased urine
volume, concomitant bladder mass increase and decreased osmolality)
in the male rat. However, when the starch component of the 5%
saccharin-containing diet was replaced with an equivalent amount of
glucose, the effect on caecal enlargement was the same, leading the
authors to conclude that in addition to inhibiting carbohydrate
digestion, 5% sodium saccharin in the diet of rats also inhibited
intestinal transport of glucose. A diet low in carbohydrates (3%
sucrose) resulted in a smaller increase in caecal volume compared
with diets containing various forms of carbohydrates at a level of
65%. It abolished the increase in relative urine volume and bladder
mass noted in the carbohydrate-fed groups even though low-
carbohydrate groups had a comparable increase in water intake. The
authors concluded that the responses of urinary parameters in rats
ingesting high doses of sodium saccharin were dependent on the
effects of sodium saccharin on carbohydrate metabolism and glucose
transport from the intestine. However, they did not take into
consideration the effects of the high concentrations of cellulose
and fat in the low-carbohydrate diet on the absorption of water from
the GI tract. They also did not comment on the fact that absorption
of ingested saccharin was markedly decreased in rats on the low
carbohydrate diet compared with the 65% carbohydrate diets; only
31% of the ingested dose of saccharin was excreted in the urine
compared with 67-83% in the carbohydrate diets (Anderson et al.
Measurement of the caecal contents of the rat has shown that
sodium saccharin ingestion increases the total protein content of
the caecum (Sims and Renwick 1985) and produces dose-related
increases in the urinary excretion of the bacterial amino acid
metabolites, indican and p-cresol, which are known to have
promoting or co-carcinogenic properties (Lawrie et al. 1985;
Lawrie and Renwick 1987). The toxicological significance of these
abnormal metabolic profiles is uncertain.
Comparison of the extent of hyperplasia of the bladder
epithelium in rats fed diets containing 5% sodium saccharin and/or
1.5% indole has demonstrated that indole per se does not
contribute significantly to hyperplasia in the urinary bladder
(Anderson et al. 1989).
Administration of 1 g/day of sodium saccharin to humans did not
alter the urinary excretion of these bacterial amino acid
metabolites (Lawrie and Renwick 1987).
A mechanism of action involving enhanced microbial activity in
the gut resulting from excess undigested carbohydrates and protein
in saccharin-treated rats was not apparent.
2.3 Observations in humans
Epidemiology studies on the possible association between
saccharin ingestion and bladder cancer in humans covering the period
up to 1983 were reviewed. The studies and conclusions included in
this review were mentioned in the previous monograph addendum (Annex
1, reference 67), including a meta-analysis of 8 of the studies
which concluded that the relative risk associated with ingestion of
saccharin and subsequent development of bladder cancer was close to
one for males, females or both sexes combined (Morgan and Wong
Since the review of 1985, there have been additional
epidemiology studies published which considered the potential effect
of saccharin on the urinary bladder. A novel study using autopsy
specimens reported on the histological changes in sections from the
urinary bladder in humans in which the numbers of cell rows and the
presence and extent of cells with atypical nuclei were recorded. A
total of 6503 sections from 282 patients were examined. No
relationship was found between the changes in the bladder epithelium
and the use of artificial sweeteners in general (Auerbach and
Another important study was published by the group which had
previously reported a significantly increased saccharin-related risk
for males (odds ratio 1.6) but not females (Howe et al., 1977).
The more recent case-control study used 826 histologically-verified
cases of bladder cancer (compared with 480 men and 152 women in the
earlier study). The relative risk for the use of a number of
artificial sweeteners, including saccharin, did not suggest an
association with bladder cancer in either males or females (Risch
et al. 1988).
Two studies attempted to identify the principal risk factors
for bladder cancer in Spain using 406 patients with bladder cancer
(353 males and 53 females) and age-matched controls from the same
hospital who did not have any malignant disease. Consumption of
wine with "gaseosa", which contains saccharin and cyclamate, was
associated with an enhanced risk of bladder cancer in males although
there was no association between alcohol ingestion and bladder
cancer (after stratification for smoking). The authors did point
out, however, that the wine consumed with gaseosa was typically of
low quality and contained large amounts of impurities.
Consequently, the low quality of the wine was an additional risk
factor (Bravo and Del Rey-Calero 1987).
In a related paper, discriminant analysis was applied to the
data obtained from the same patients (not presented) in order to
identify and rank factors increasing the risk of bladder cancer.
Use of artificially-sweetened beverages ranked fourth and use of
artificial sweeteners themselves ranked eighth. The number of
cigarettes smoked was the most important factor. Saccharin was not
specifically mentioned (Bravo et al. 1987).
A small case-control study (194 bladder cancer patients and the
same number of age- and sex-matched controls) conducted in Turkey
reported a statistically significant association (p<0.05) between
the use of artificial sweeteners and the development of bladder
cancer. The authors did not distinguish between the use of specific
sweeteners and only 19 cases and 8 controls reported using these
substances. In addition, there was no investigation of the possible
influence of confounding factors (Akdas et al. 1990).
Two population-based case-control studies in the U.S.A.
concluded that there was no link between consumption of artificial
sweeteners and bladder cancer. One study was conducted with 173
female patients with bladder cancer and had only two categories of
use of artificial sweeteners, that being 100 times or more life-time
use of artificially-sweetened beverages or tabletop sweeteners or
less than 100 times use (Piper et al. 1986).
The second study investigated the potential of increased volume
of fluid intake or of specific fluids, including artificially-
sweetened beverages, to increase the risk of bladder cancer. No
association was found between the volume of intake of artificially-
sweetened beverages and bladder cancer (Slattery et al. 1988).
A case-control study with 117 patients apparently showed no
association between saccharin consumption and bladder cancer (the
data were not presented) (Iscovitch et al. 1987). Although this
study was small compared with some previous investigations, it was
of sufficient power to show that cigarette smoking was a major risk
An update of the earlier review of Morgan and Wong (1985) in
which most of the above-mentioned studies were analyzed, was
provided. In addition, they added two of these studies to the
meta-analysis performed previously. The conclusions from this
meta-analysis were the same as those in the previous paper: the
relative risk from the combined data of 15 studies indicated that
there was no association between ingestion of saccharin and
development of bladder cancer (Elcock and Morgan 1992).
A study which investigated the correlation between the urinary
excretion of the microbial amino acid metabolites indican,
p-cresol and phenol (which have been shown to be co-carcinogenic
or promoters) and bladder cancer was conducted in thirty-two
patients with histologically-confirmed carcinoma of the urinary
bladder and a similar number of matched controls. There was wide
variability between individuals in the excretion of these
metabolites, but no difference was detected between the two groups
(Renwick et al. 1988).
An independent assessment of the bladder histopathology data
from the most recent two-generation feeding study in rats that was
reviewed at the twenty-eighth meeting revealed the presence of
transitional cell papillomas and carcinomas in the control group.
This assessment reduced concern over setting the NOEL at a dose
where tumours were observed and it eradicated the statistical
significance of the increase in tumour incidence at the 3% dietary
level. Application of a dose-response model to the carcinogenicity
data suggested a threshold for carcinogenesis close to 3% saccharin
in the diet. This is the dietary level at which saturation of renal
tubular secretion occurs in the rat and anaemia and other
biochemical changes occur in weanling rats. In this study, the
absolute and relative weights of the urinary bladder of treated rats
were significantly higher than those of controls when sodium
saccharin was included in the diet at levels of 3% or higher.
The rat is the only species that has been reported to show an
increase in the incidence of bladder tumours at high dietary
concentrations of sodium saccharin in a 2-generation study. Apart
from mice, studies in other species have not included neonatal
exposure to saccharin at levels above the maximum tolerated dose.
The Committee concluded from the long-term feeding studies that the
dose-related carcinogenic activity of sodium saccharin on the
urinary bladder was specific to the male rat and that exposure
during the neonatal period was critical for the subsequent
development of these tumours in the absence of an initiator or
stimulus such as freeze ulceration. The critical events during the
neonatal phase that lead to an increase in the population of
initiated cells have not been identified.
The Committee considered the genotoxic potential of saccharin
on the basis of its physicochemical properties and results from
in vitro and in vivo assays. At physiological pH, saccharin
exists almost exclusively as the anion. As such, the parent
compound does not resemble an electrophilic chemical carcinogen that
would bind to DNA, nor has it been shown to bind to DNA in vivo.
Because it is not metabolized, it is not converted to an active
metabolite. On the other hand, sodium saccharin has exhibited
clastogenic activity in a number of in vivo and in vitro
genotoxicity assays. Since high concentrations of sodium saccharin
were used in these assays, it has been suggested that the
clastogenic activity could be attributable to ionic imbalances at
the chromosomal level at high concentrations. The clastogenic
activity is also in disagreement with the results of the long-term
studies and tumour-initiation promotion studies with sodium
The conditions required for the hyperplastic and tumour-
promoting activities of high dietary concentrations of saccharin
(usually 5% or higher) on the urothelium in the male rat are an
increased urinary concentration of sodium ion and an elevated pH.
The response does not appear to be specific to saccharin, since high
dietary concentrations of other organic anions have been shown to
promote bladder carcinogenesis and induce urothelial hyperplasia
under the same conditions. The differences in tumour-promoting
activities observed between organic acids and their sodium salts
were unrelated to the urinary concentration of the parent organic
The Committee was not convinced that the available evidence
implicated alpha2u-globulin in bladder carcinogenesis. The
Committee also noted that a proposed mechanism of action involving
enhanced microbial activities in the gut, resulting from excess
undigested carbohydrates and protein in rats administered saccharin
in the diet had been investigated without any conclusive evidence.
The epidemiological studies on saccharin did not show any
evidence that saccharin ingestion increases the incidence of bladder
cancer in human populations.
The Committee accepted that, on the basis of data reviewed to
date, it would be inappropriate to consider the bladder tumours
induced in male rats by sodium saccharin to be relevant to the
assessment of a toxicological hazard to humans.
In re-assessing saccharin, the Committee considered that the 1%
dietary level in the most recent 2-generation feeding study in rats,
equivalent to 500 mg/kg bw/day, was appropriate for establishing an
intake causing no relevant toxicological effects. This was based on
the observation that, although dose levels of up to 7.5% sodium
saccharin in the diet had no adverse effect on survival, the animals
demonstrated a marked disturbance of homoeostasis at levels of 3%
and higher. In particular, persistent dose-related decreases in
body weight gain in the presence of increased food consumption are
indicative of decreased biological performance and were probably
related to the inhibitory effects of saccharin on carbohydrate and
protein digestion. A no-effect level of 500 mg/kg bw/day was also
observed in a long-term toxicity study in monkeys reviewed at the
twenty-sixth meeting (Annex 1, reference 60).
The Committee allocated a group ADI of 0-5 mg/kg bw/dy to
saccharin and its calcium, potassium, and sodium salts, based on the
NOEL of 500 mg/kg bw/day in the 2-generation long-term feeding study
in rats and a safety factor of 100.
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