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    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

    1  EXPLANATION

         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
    monograph addendum.

    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 g/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 g/ml (Sweatman  et al. 1981).

    2.1.2  Biotransformation

         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
    1985).

         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
    1991b).

    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.
    1991).

         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.
    1990, 1991).

         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
                                                            of saccharin
                                                                                                                         

    Cell mutation/ouabain      Human RSa cells              10 - 22.5 mg/ml         Positive      Suzuki and Suzuki 1988
    resistance

    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 g/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.
    1988).

         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 g/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 g/ml), none of
    the foci produced by MNU (25 g/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
    g/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 g/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 g/ml), but not sodium saccharin (25 g/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.

    2.2.5.1  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 (0.60.2%), the potassium
    salt resulted in a weak, but significant, effect (0.20.1%), the
    calcium salt resulted in a questionable and non-significant effect
    (0.10.1%), while the acid form (0.070.04%) gave a result similar
    to controls (0.060.04%).  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 mol/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.,
    1987).

         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).

    2.2.5.2  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.
    1988).

         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.

    2.2.5.3  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
    carcinogenicity.

    2.2.5.4  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
    1992).

         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.
    1992a).

         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.
    1991b).

         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
    identified.

    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.
    1987a).

         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
    1985).

         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
    Garfinkel 1989).

         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
    factor.

         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).

    3.  COMMENTS

         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
    saccharin.

         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
    molecule.

         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.

    4.  EVALUATION

         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.

    5.  REFERENCES

    AKDAS, A., KIRKALI, Z. & BILIR, N. (1990).  Epidemiological
    case-control study on the etiology of bladder cancer in Turkey. 
     Eur. Urol., 17, 23-26.

    ANDERSON, R.L., FRANCIS, W.R. & LEFEVER, F.R. (1987a).  Effects of
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    See Also:
       Toxicological Abbreviations