IPCS INCHEM Home

    SACCHARIN, CALCIUM, POTASSIUM AND SODIUM SALTS*

    Explanation

         Saccharin was evaluated by the Joint Expert Committee on Food
    Additives in 1967, 1974, 1977, 1980 and 1982 (Annex I, Refs. 14, 15,
    35, 44, 53, 59, and 60). In 1977, the Committee changed the ADI from
    5 mg/kg to a temporary ADI of 2.5 mg/kg and withdrew the conditional
    ADI of 15 mg/kg for dietetic purposes only. The decision to reduce the
    ADI and to restrict the use of saccharin was based primarily on the
    results of animals studies which indicated that excessive and
    long-term ingestion of saccharin was potentially a carcinogenic hazard
    for humans. At the 1980 and 1982 meetings, the temporary ADI of
    2.5 mg/kg was extended pending the completion of current
    investigations, including a long-term feeding study in rats and
    epidemiological studies. Since the previous evaluation, additional
    data has become available and is summarized and discussed in the
    following monograph addendum.

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

    Absorption

         Previous studies have shown that the absorption of ingested
    saccharin in animals and man is rapid and this is confirmed by
    observations that the peak plasma concentration occurs soon after oral
    administration to rats (Matthews et al., 1973; Sweatman & Renwick,
    1980) and to man (Colburn et al., 1981; Sweatman et al., 1981;
    Pantarotto et al., 1981a,b). The presence of food in the gut was
    associated with a reduced initial peak plasma concentration in animals
    (Matthews et al., 1973; Sweatman & Renwick 1980) and in man (Sweatman
    et al., 1981).

    Distribution

         Recent studies on the distribution of saccharin have given
    attention to the nature and amounts of radioactivity in the bladder
    tissue after administration of radiolabelled saccharin and on
    concentrations in this tissue during chronic intake.

              

    *    Monograph addendum

         In a recent two-generation study (Sweatman & Renwick, 1982) using
    3H-saccharin, it was shown that there was a slower decrease in the
    saccharin content of fetal tissues than of maternal tissue, and in
    particular, the concentration of saccharin in the fetal bladder wall
    decreased relatively slowly during a 48h period following a single
    oral dose to the dam. Despite this, the steady state concentration of
    saccharin in the liver and kidneys of fetuses from mothers fed a 5%
    saccharin diet were lower than the maternal values while the
    concentrations in the fetal bladder were similar or slightly higher.
    It was concluded that there was no evidence of excessive accumulation
    in the bladder wall or other tissues of male rats during in utero
    exposure or during lactation which could explain sex and generation
    specificity of the tumorigenic response.

    Excretion

         The saturation of renal tubular secretion of saccharin in rats
    fed high dietary levels was previously demonstrated by comparison of
    plasma concentrations following intravenous infusion and chronic
    dietary intake (Sweatman & Renwick, 1980) and, more recently, Sims and
    Renwick (1983) found a marked decrease in renal clearance rates in
    rats with high plasma concentrations (200-300 g/ml) of saccharin.

    Effects on enzymes and other biochemical parameters

         Sodium saccharin, at concentrations similar to those in urine of
    rats fed 1-5% sodium saccharin in their diet, markedly inhibited
    urease and the proteases pepsin, thermolysin and papain (Lok et al.,
    1982) and trypsin (Sims & Renwick, 1983a). Inhibition of proteolysis
    in vivo was the probable cause of the high levels of protein and
    tryptophan in the caeca of rats fed saccharin-containing diets (Sims &
    Renwick, 1983). In this latter study, the metabolism of tryptophan by
    the caecal bacteria was altered with increase degradation to indole
    and indolelactic acid.

         Increased metabolism of protein to tryptophan and indole in the
    caecum occurred throughout a two-generation rat feeding study and the
    lactating dams showed increased excretion of indican (the main urinary
    metabolite of indole) via the milk; the pups also showed caecal
    enlargements, increased protein and tryptophan in the caecum and an
    increased excretion of indican immediately on weaning on to a
    saccharing-containing diet. These changes persisted throughout life
    as seen by subsequent analysis of urine from rats in the IRDC
    carcinogenicity study (see special studies on carcinogenicity) at 13,
    18, 24, 28 m (Renwick, 1983).

         In a study of 15 human volunteers (Renwick, 1983) administration
    of saccharin (1 g/d for 1 month) did not significantly increase the
    daily excretion of indican in urine compared with the pre- and
    post-administration control periods.

    TOXICOLOGICAL STUDIES

    Special studies on renal function

         Renal slices from rats fed diets containing 5% or 7.5% saccharin
    showed a reduced accumulation of para-animohippurate (PAH) in vitro
    but feeding these diets did not result in a reduced renal clearance of
    PAH in vivo (Berndt et al., 1981).

         The renal clearance of endogenous indican in saccharin treated
    rats showed a highly significant inverse relationship to the plasma
    concentration of saccharin (Sims & Renwick, 1983).

    Special studies on urine volume and composition

         Rats fed high dietary levels of saccharin showed an increase in
    fluid intake and in urine volume which was accompanied by a decrease
    in osmolality (Anderson, 1979; West & Jackson, 1981; Demers et al.,
    1981; Berndt et al., 1981). Dose related decreased osmolality and
    increased urine volume showed a strong correlation to the occurrence
    of bladder tumours in the IRDC carcinogenicity study (see special
    studies on carcinogenicity). These changes were primarily observed at
    dietary concentrations of 3% saccharin and above. The increased daily
    urinary volume was accompanied by both an increased volume per
    micturition and an increased frequency of micturition. Saccharin-fed
    rats showed a greater maximal distension of the urinary bladder
    (Renwick & Sims, 1983) and these authors concluded that the increased
    bladder distension would increase the possibility of interaction
    between the epithelium and endogenous urinary metabolites, especially
    during hours of daylight.

         Administration of saccharin at a dose of 1 g/d to human
    volunteers for 1 m did not affect urine volume when compared to pre-
    and post-treatment control periods (Roberts & Renwick, unpublished
    results).

         The effects of sodium saccharin on mineral and water balance and
    a number of related parameters were studied over a 10-day period in
    seven month old rats (Schoeing & Anderson, 1983). The study included
    eight groups, each consisting of 10 males and 10 females. Rats in four
    of the groups were from the second generation, the parental generation
    having been exposed to dietary concentrations of 1.0, 3.0, 5.0, or
    7.5% saccharin prior to and during gestation and lactation; the second
    generation weanlings received the corresponding diets. The treatment
    in two other groups was modified so that rats in one group were
    exposed only in utero (via dams fed diets containing 5% sodium
    saccharin) while exposure of the second group was started at birth
    (via lactation dams fed similar diets) and continued at a dietary
    saccharin concentration of 5%. The purpose of these modifications was
    to evaluate the role of in utero exposure on the study parameters. A

    group of second generation rats fed diets containing 5% soldium
    hippurate was included to evaluate the specificity of sodium saccharin
    and/or the effect of sodium ion on the study parameter. A group of
    untreated animals served as controls. At dietary sodium saccharin
    concentrations >1%, increases in water consumption and urine volume
    were noted. At dietary concentrations >3.0% decreased urine
    osmolality, changes in water and mineral balance, increased mass of
    the caecum and bladder, and increases in bladder tissue mineral
    concentrations were observed; the latter effect was noted only in male
    rats. The evaluation of these parameters in rats with and without
    in utero exposure indicated that in utero exposure played little
    or no role in the occurrence or severity of these changes.
    Qualitatively similar, but quantitatively less severe changes were
    observed in rats fed sodium hippurate.

    Special studies on caecal enlargement and stool hydration

         In an attempt to determine why dietary sodium saccharin causes
    caecal enlargement and increased stool hydration, Anderson (1983)
    analyzed stools from rats fed diets containing 1, 3, 5, or 7.5%
    saccharin. Saccharin ingestion resulted in a small increase in stool
    ash but no change in lipid or non-saccharin nitrogen concentrations
    (mg/g dry stool). Saccharin treatment also resulted in a dose-
    dependent increase in the stool content of a carbohydrate soluble in
    1M-NaOH. The author suggested that the source of the stool
    polysaccharide was either undigested dietary polysaccharide or a
    product of intestinal microbial synthesis and that the hygroscopic
    carbohydrate together with the high stool saccharin content caused
    caecal enlargement and increased stool hydration. In this context,
    Shibata et al., (1983) showed that a strain of Streptococcus
    obtained from the rat caecum produced an extra-cellular hygroscopic
    glucan when grown in the presence of sucrose.

    Special studies on effects on the bladder epithelium

         Sodium saccharin was fed to male F344 rats at dietary levels of
    0, 0.1, 0.5, 1.0, 2.5 or 5% for 10 weeks. Food consumption and body
    weight gain were similar in all groups and no gross signs of toxicity
    were observed. Sodium saccharin induced a dose-dependent proliferation
    of the urinary bladder mucosa as assessed by autoradiography and
    scanning electron microscopy. All rats in all groups had cells with
    ropy microridges and uniform microvilli; sodium saccharin at dietary
    levels >1% increased the number and size of these foci. In addition,
    pleomorphic microvilli were observed at the two highest dose levels
    but not in controls (Murasaki & Cohen, 1981).

         Strain and species differences in the response of the urinary
    bladder to sodium saccharin were observed by Fukushima et al. (1983a).
    Male ACI, Wistar, F344 and Sprague-Dawley rats were given a diet
    containing 5% sodium saccharin for 52 weeks. In ACI rats, sodium

    saccharin induced not only preneoplastic lesions but also bladder
    tumours; in other strains it did not. The urinary bladder of ACI rats
    had the most marked lesions under scanning electron microscopy, with
    less marked changes in Wistar and F344 rats; Sprague-Dawley rats were
    resistant to these changes.

         Male F344 rats, B6C3F1 mice, Syrian hamsters and Hartley guinea
    pigs were given 5% sodium saccharin in the diet for 20 weeks. Animals
    from each group and respective controls were sacrificed at 0, 4, 8,
    12, 6 and 20 weeks after commencement of feeding. The rats developed
    urinary bladder lesions as detected by light and electron microscopy
    and increased DNA synthesis of the urinary bladder epithelium was
    detected by autoradiography.  Mice, hamsters and guinea pigs were
    resistant to sodium saccharin.

         Male and female Sprague-Dawley rats eight weeks of age were
    given saccharin according to the standard IRDC protocol and the
    effects on endogenous mitotic activity in the bladder assessed by
    autoradiographic measurement of the thymidine labelling index (Tsing,
    1983). In contrast to earlier reports (Fukushima & Cohen, 1980;
    Murasaki & Cohen, 1981) no treatment related effects on the thymidine
    labelling index were observed; the index was higher in male than
    female rats. The differences between the results of these different
    studies may have been due to genetic differences between the strains
    of rat used. Reitz et al. (1983) also reported no significant
    differences in the thymidine labelling index between controls and
    treated Fisher 344 rat pups exposed to 7.5% saccharin in the diet
    in utero and subsequently up to 35 days. The thymidine labelling
    index was determined 8 days and 35 days post partum.

         The changes in membrane potential of the epithelium of the F344
    rat bladder have been measured following treatment of the animals with
    BBN or saccharin (Iamida et al., 1983). Dietary concentrations of O,
    0.04, 0.2, 1 or 5% sodium saccharin were administered but only the
    highest dose level caused a significantly higher membrane potential
    than the control group.

         Recently, El Gerzawi et al. (1982) obtained normal human bladder 
    tissue and studies the effects of N-methyl-N-nitroso-urea (MNU) and
    saccharin on the histology of the epithelium in long-term explant
    cultures.  In MNU-treated cultures, a dose-response was observed.
    Single doses of 1-100 g MNU/ml induced a typical hyperplasia, however
    the changes reverted to a single or double cell layer as seen in
    controls. In contrast, after multiple doses of MNU the hyperplastic
    changes persisted. The doses of MNU in the presence of saccharin
    gave cellular changes similar to those seen with multiple doses of
    MNU alone although the nuclei appeared more pleomorphic and
    hyperchromatic. Continuous exposure of the explants to saccharin alone
    did not result in any changes from the controls.

    Special studies on food consumption patterns

         Utilizing a protocol recommended for two-generation bioassays,
    Reitz et al (1983) reported that when rats were given a diet
    containing 7.5% sodium saccharin through gestation and lactation, the
    very young animals received considerably more sodium saccharin on a
    mg/kg body weight per day basis than did adults consuming the same
    diet; the young animals suffered weight depression and early mortality
    when exposed to a dietary concentration which was well tolerated by
    adults. These authors concluded that failure to maintain the dose of
    the MTDS of 5000-6000 mg/kg body weight per day throughout a
    two-generation study would compromise its usefulness in the
    formulation of human risk estimates.

         Three groups of 5 Rhesus monkeys were used in a study designed to
    determine the maximum amount of sodium saccharin which monkeys would
    voluntarily consume and/or tolerate (Westland & Helton, 1983); sodium
    saccharin was incorporated into the dry diet of one group and the
    drinking water of a second group with the third group serving as
    controls. An increasing dose regimen in which the concentration of
    sodium saccharin in the diet or drinking water was doubled every three
    days was employed. Concentrations between 0.125 and 8.0% were
    evaluated in the diet, and between 0.015 and 0.48% in the drinking
    water. Very little rejection of either diet or drinking water was
    observed but severe diarrhoea precluded further treatment when the
    concentration of sodium saccharin reached 8.0% (approximately 
    1600 mg/kg per day) in the diet and 0.48% (between 900 and 2400 mg/kg 
    per day) in the drinking water. An increase in fluid intake occurred 
    in monkeys in both treatment groups. In addition, an increase in urine
    volume and a decrease in urine osmolality were seen in monkeys given
    sodium saccharin in drinking water. No effects on body weight, food
    consumption or urine pH were observed in either group. All animals
    recovered rapidly after being returned to untreated diet or water.

    Special studies on mutagenicity of impurities in saccharin

         The known impurities in sodium saccharin produced by the Maumee
    (Sherwin-Williams) and the Remsen-Fahlberg processes have been
    tabulated, and saccharin produced by the former process was analysed
    to determine the concentration and identities of impurities (Riggin
    & Kinzer, 1983). Most of the contaminants were found to be derived
    from the polythene bags in which the saccharin was packed.
    N-methylsaccharin (0.15 ppm) and methyl anthranilate (0.05 ppm) were
    the predominant impurities. The major polar impurities in the Maumee
    product were identified as 5-, 6-, and 7-aminosaccharin which were
    present at a combined level of approximately 150 ppm (Radford et al.,
    1983); these polar metabolites occurred to only a minor extent in
    Remsen-Fahlberg saccharin (Wolf & Voigt, 1983).

         Recently, an evaluation of the mutagenic activity of a mixture
    of the major polar impurities of Maumee saccharin and of 5- and
    6-aminosaccharins was carried out using the Ames Salmonella assay;
    these compounds were found to be non-mutagenic with or without
    metabolic activation by S9-mix (Litton Bionetics, Inc., 1983,a,b,c).
    Similarly, Riggin et al. (1983) examined the mutagenic activity of
    solvent extracts of specific manufacturing lots of saccharin produced
    by the Sherwin-Williams process. All the individual components
    identified were found to be non-mutagenic in the Ames assay. A weak
    mutagenic activity was associated with chloroform extracts from one
    lot of saccharin representing less than 1.5 ppm of the sample but was
    not attributable to a single component. The possibility of artefact
    formation from solvent interaction with impurities could not be
    ruled out. The authors concluded that these impurities are of no
    toxicological significance in animal feeding studies.

    Special studies mutagenicity of saccharin

         Saccharin was classified as highly mutagenic in an in vivo
    mammalian spot test for detection of genetic alterations in mouse
    embryo pigment cells (Mahon & Dawson, 1982). Offspring heterozygous
    for several coat-colour genes were exposed in utero by
    administration of saccharin to the dams at intra-gastric doses of
    0.075, 0.75, 1.5, 3.0, 5.0, or 7.5 g/kg body weight on days 8, 9, or
    10 of pregnancy. The presence of colour spots on the cost of the
    offspring was taken as indicative of expression of a recessive
    phenotype resulting from alteration of loss of a wild-type allele from
    a prospective pigment cell. The frequency of saccharin-treated mice
    (all dose levels) with colour spots was 3.6% compared to a control
    frequency of 0.9% (P = 1  10-6) but there was no significant
    variation in frequency due to dose over the wide dose range used. The
    lack of a proportional dose response was acknowledged to be unusual in
    this test.

         In contrast, Fahrig (1982) categorized saccharin (Remsen-
    Fahlberg, containing 27 ppm OTS) as non-mutagenic in the mammalian
    spot test. In this study, saccharin (1 g/kg body weight) was
    administered by i.p. injection on day 10 of pregnancy. Only one spot
    of genetic relevance was found among 701 saccharin treated offspring
    and this did not differ from the spontaneous frequency. The effect of
    1 g/OTS kg body weight given orally was also evaluated in three
    replicate tests, only one of which gave statistically significant
    positive results. A clear classification of OTS as mutagenic or
    non-mutagenic in this test was not possible.

         The genotoxic potential of sodium saccharin and of 1-naphthalene
    sulphonic acid was evaluated in the rat hepatocyte unscheduled DNA
    synthesis assay at concentrations ranging from 1  10-4 to 1  10-1
    M. Both materials were toxic to hepatocyte cultures at 3.16  103 to
    1 xz 10-1 M.  Decreasing concentrations of sodium saccharin or

    1-naphthalene sulphonic acid resulted in decreased toxicity and the
    culture exposed to 1  10-3 to 1  10-4 M concentrations resembled
    negative controls. Neither compound elicited significant unscheduled
    DNA synthesis at any of the concentrations tested (Reitz & Medrala,
    1983).

    Special studies on carcinogenicity-promoting or co-carcinogenic effects

         The promoting effects of sodium saccharin and of phenobarbital
    (PB) on all organs of rats were studied after initiation with
    N-mitrosomethylurea (NMU) (Tsuda et al., 1983). Three groups of 25
    male F344 rats were given 20 mg NMU/kg body weight i.p. twice a week
    for 4 weeks, then given a diet containing 0.05% PB or 5% sodium
    saccharin for the next 32 weeks. The animals were then killed, a
    complete necropsy performed and sections of all tissues were stained
    (H&E) and examined microscopically. The results indicated that PB
    promoted the induction of neoplastic or preneoplastic changes in the
    thyroid and liver but that sodium saccharin acted exclusively as a
    promoter in the urinary bladder. There were significant increases in
    the incidence and number of PN-hyperplastic changes (P <0.01) in the
    bladders of animals given NMU + sodium saccharin. No papillomas were
    observed in the bladders of any rats in this study.

         The possibility of sodium saccharin acting as a co-carcinogen was
    studies by co-administration of sodium saccharin and N-(4-(5-nitro-2-
    furyl)-2-thiazolyl)-formamide (FANFT) at dietary levels of 5% and
    0.005% respectively to male Fisher rats for 2 years. The effects of
    L-tryptophan (2% of the diet) were also studied alone or
    co-administered with sodium saccharin. Five of the sixteen rats given
    sodium saccharin plus FANFT developed bladder tumours whereas none of
    the animals given FANFT, L-tryptophan or sodium saccharin alone,
    sodium saccharin plus tryptophan or control diets, developed bladder
    tumours. Two of the rats receiving only FANFT developed papillary and
    nodular hyperplasia. The results were stated to indicated that sodium
    saccharin had no-carcinogenic activity when given simultaneously with
    FANFT (Murasaki & Cohen, 1983a).

         Rats were fed diets containing 0 or 5% sodium saccharin
    immediately, 2 weeks or 8 weeks after freeze ulceration of the urinary
    bladder and the effects studied by light and scanning electron
    microscopy and by measurement of the thymidine labelling index. Sodium
    saccharin prolonged the regenerative hyperplastic changes following
    ulceration and maintained an increased proliferative rate in the
    epithelium. Delaying saccharin administration for 8 weeks after
    ulceration still resulted in nodular and papillary lesions, surface
    abnormalities detected by scanning electron microscopy and an
    increased labelling index. These changes were thought to contribute to
    the eventual induction of bladder neoplasma in rats fed sodium
    saccharin following ulceration (Murasaki & Cohen, 1983b).

         Recent studies by Nakanishi et al. (1982) were conducted to
    determine if a 4 week pre-treatment with N-butyl-N-(4-hydroxybutyl)
    nitrosamine (BBN) or N-2fluorenylacetamide (2-FAA) followed by 5%
    saccharin or 0.05% phenobarbital (PB) in the diet for 32 weeks induced
    or enhanced liver and/or bladder tumours. Male F344 rats were
    pretreated with 0.02% 2-FAA or 0.01% BBN in the drinking water. The
    results indicated that, while 2-FAA and BBN have tumour-initiating
    effects in both the liver and urinary bladder, the promoting effects
    of saccharin and PB are organ-specific. Similar results were reported
    by Tsuda et al. (1983).

         The effect of partial cystectomy on the occurrence of
    pre-neoplastic lesions, papillary or nodular hyperplasia
    (PN hyperpl.asia) of the bladder in male F344 rats was studied in an
    experiment in which bladder carcinogens and promoters were given to
    the rats after initiation with BBN. The bladder carcinogens tested
    were N-ethyl-N-(4-hydroxybutyl) nitrosamine (EHBN) at a level of 0.01%
    in drinking water or FANFT at a dietary level of 0.2%. The promoters
    used were sodium saccharin (5%), sodium cyclamate (2.5%), or
    DL-tryptophan (2%) in the diet. Partial cystectomy significantly
    decreased the occurrence of PN hyperplasia in rats treated with EHBN
    and tended to inhibit that in rats given saccharin or tryptophan i.e.,
    partial cystectomy inhibited rather than enhanced the induction of PN
    hyperplasia (Fukushima et al., 1982).

         Fukushima et al. (1983b) studied the promoting effects of various
    chemicals on bladder carcinogenesis in 22 groups of 30 males F344 rats
    after initiation by BBN. The rats were initially given 0.01% BBN rats
    in the drinking water for 4 weeks and then the test compounds in the
    diet for 34 weeks. The test compounds used were: sodium saccharin (0.5
    and 5%) sodium ascorbate (5%), calcium carbonate (5%), DL-tryptophan
    (5%) allopurinol (0.02%), acetazolamide (0.35%), quercetin (5%),
    sodium hippurate (5%) and vitamin D (0.002%). Each of these compounds
    was given to two groups of 30 rats. Effects were judged by measuring
    the formation of preneoplastic lesions - papillary or nodular
    hyperplasia (PN hyperplasia) of the urinary bladder. Administration of
    5% but not 0.5% sodium saccharin in the diet significantly increased
    the incidence and extent of PN hyperplasia; sodium ascorbate,
    DL-tryptophan and allopurinol also increased the extent of PN
    hyperplasia but the other test compounds did not at the dietary
    concentration used. The results with sodium saccharin and
    DL-tryptophan were consistent with earlier findings and the results
    with sodium ascorbate and allopurinol suggest that these compounds
    have promoting activities in urinary bladder carcinogenesis in the
    rat. No correlation was found between the extent of crystalluria and
    promotion of pre-neoplastic lesions.

         In a two-stage bladder carcinogenesis study, Ito et al. (1983a)
    evaluated the promoting effects of 16 test chemicalsby their ability
    to induce PN hyperplasia in F344 rats. Male rats were given 0.01% BBN

    in drinking water for four weeks followed by one of the test compounds
    for 32-34 weeks. The dose response of saccharin was also studied in
    rats of both sexes at dietary concentrations of 0, 0.04, 0.2, 1.0 and
    5% for 32 weeks after BBN treatment. Dose-response curves showed
    enhanced hyperplasic responses in both sexes given 0.2 to 5%
    saccharin. Ito et al. (1983a) also studied the organ specificities of
    phenobarbital or saccharin after initiation with BBN or 2AAF; the
    promoting effects were found to be organ specific. Similar findings
    were reported in other studies (Nakanishi et al., 1982; Tsuda et al.,
    1983).

         A number of tumour promoters in the two-stage mouse-skin
    carcinogenesis system are known to be reversible inhibitors of nerve
    growth factor-induced neurite out-growth while their non-promoting
    structural congeners are not.  A 50 mM concentration of Maumee sodium
    saccharin inhibited neurite out-growth; the inhibition was completely
    and rapidly abolished by washing out the saccharin. Saccharin also
    inhibited binding of 125I-nerve growth factor in embryonic chick
    sensory ganglia cells in a concentration dependent manner (Ishii,
    1982). It was postulated that alteration of cellular differentiation
    by tumour promoters may result from interactions with receptor systems
    that regulate cellular function.

    Special studies on carcinogenicity

    Rat

         A two-generation carcinogenicity study has been performed with
    the primary objective of investigating the dose-response curvre for
    urinary bladder tumours in male Charles River CD rats. The study was
    also designed to evaluate the role of in utero exposure, the
    specificity of sodium saccharin and the role of excess sodium in the
    occurrence of urinary bladder tumours.

         First generation (Fo) parental animals were given diets
    containing 0, 1.0, 3.0, 4.0, 5.0, 6.25 and 7.5% sodium saccharin from
    approximately six weeks of age for approximately 4 1/2 months. During
    this time, the animals were mated (one male to two females) commencing
    with 110-114 days old and the females were allowed the nurse the
    offspring for 21 days. When the second generation (F1) offspring were
    between 28 and 38 days old, second generation male rats were randomly
    selected from each treatment group for the chronic phase of the
    bioassay. At this point, all Fo male and female rats, all F1 female
    rats and F1 males not selected for the carcinogenicity study were
    removed from the study. F1 males selected for the chronic phase were
    maintained on the same diet which their parents received for a period
    of up to 30 months.

         Two further treatment groups were also included to study the
    possible role in in utero exposure in the production of urinary
    bladder tumours. The Fo animals in one group were fed sodium
    saccharin at a dietary concentration of 5% only during mating and
    gestation. After parturition, the dams were fed control diet and
    selected F1 males were continued on the control diet for a period of
    30 months. This group was designated "5% saccharin through gestation".
    In the second group, Fo animals were maintained on control diet until
    parturition after which the dams were placed on a sodium saccharin
    test diet beginning at 1% and increasing to 5% during lactation.
    Selected F1 males from these parents were fed sodium saccharin at a
    dietary concentration of 5% for 30 months. This group was designated
    "5% saccharin following gestation".

         A third treatment group was included to study the effect of
    excess sodium and to determine the specificity of high doses of
    saccharin to the occurrence of bladder tumours. Animals in this group
    were fed a diet containing 5% (reducing to 3%) sodium hippurate
    through two generations. This compound was selected because of its
    similarity to saccharin in physical, chemical and pharmacokinetic
    properties, e.g. both are sodium salts of organic acids of almost
    identical molecular weight and are filtered and actively secreted into
    urine by the kidney.

         Selected F1 males from each of the groups were allocated to the
    second generation long-term study using the following unbalanced
    design:

    F1 treatment group                           Number of F1 males
                                                                   

          untreated control                             350
    1.0%  sodium saccharin                              700
    3.0%  sodium saccharin                              500
    4.0%  sodium saccharin                              200
    5.0%  sodium saccharin                              125
    6.25% sodium saccharin                              125
    7.5%  sodium saccharin                              125
    5.0%  sodium saccharin through gestation            125
    5.0%  sodium saccharin following gestation          125
    5.0/3.0% sodium hippurate                           125

         The F1 animals were observed twice daily for signs of toxicity,
    behavioural changes and survival. Individual body weights and food
    consumption were measured weekly during the first 13 weeks and once
    every two weeks thereafter. Urinanalyses were conducted on 30
    rats/group on days 6, 30, 64, 92 and months 6, 13, 18, 24 and 29. The
    urinanalysis included urinary pH, microscopic examination, bilirubin,
    protein, glucose, ketones, urobilinogen, nitrite and occult blood.
    Osmolality was also measured on fresh urine samples on day 6, 30 and

    64, and on 24 h urine samples at each examination except day 6.
    Individual 48 h water consumption was measured on the same 30 animals
    per group for the first 13 weeks and approximately every two weeks
    thereafter.

         During the F1 phase, complete post-mortem examinations were
    performed on all animals which died during the course of the study or
    were sacrificed in extremis and on all terminally sacrificed animals.
    A complete range of tissues was fixed for possible subsequent
    examination and the urinary bladder, kidney, urethra and ureters of
    all animals were examined histologically. Gross lesions and masses,
    from all tissues, observed at autopsy were also examined
    microscopically. Examinations of the fixed urinary bladder and
    subsequent microscopic examinations of tissues were conducted in a
    blind manner.

    Results of the Fo generation and litters

         The feeding of sodium saccharin at dietary concentrations up to
    7.5% to male and female Fo rats from the post-weanling stage through
    a single reproductive cycle had no effects on behaviour or survival.
    However, at dietary concentrations <3.0% statistically significant
    (P>0.05) body weight depressions were noted. The difference from the
    untreated control group was as high as 11% in the 7.5% sodium
    saccharin treatment group. The depressions in body weight were not due
    to a decrease in food consumption or nutrient intake since the treated
    rats compensated for the non-nutritive ingredient added to their diet
    by consuming more total diet (g/rat/d) than untreated controls. At
    dietary concentrations of sodium saccharin <3% there were significant
    (P>0.05) reductions in the mean number of pups per litter.  There was
    a statistically significant increase in water intake and urine volumes
    at dietary levels <1%; a decrease in urinary pH and visible increase
    in the moisture content of faeces were observed at dietary
    concentrations <3%.

         No effects on survival or behaviour were observed through the
    first 28 to 38 days of life in the offspring receiving dietary
    concentrations of sodium saccharin up to 7.5% and the mean body
    weights at birth were also comparable to control animals. However,
    statistically significant (P>0.05) body weight depressions were noted
    in all sodium saccharin groups later during the lactation period and
    in males at 28 days of age. The difference was noted only at days 21
    and 28 in the 1.0% sodium saccharin group and was small (approximately
    2%); by comparison, the average body weight depressions in the 3.0%
    through 7.5% sodium saccharin groups were between 8% and 24% at day
    21, and between 9% and 31% at day 28. The weanling rats (28-38 day
    old) from the 5% and 7.5% sodium saccharin groups were found to be
    anaemic.

         The feeding of 5.0% sodium saccharin only during mating and
    gestation caused statistically significant depressions (P<0.05) in
    both food consumption and body weight of the Fo animals during the
    treatment period. A statistically significant (P<0.05) decrease in
    the mean number of pups born per litter was also observed which was
    comparable to the decrease noted in the dose-response group in which
    parental animals were fed 5.0% sodium saccharin continuously from the
    weaning stage. No changes were observed in the offspring during the
    first 28 to 38 days of life.

         The feeding of sodium saccharin to lactating female rats on an
    increasing dosage schedule of 1%, 3% and 5% during weeks 1, 2 and 3
    post-parturition respectively had no observable effect until the
    dietary concentration of sodium saccharin was increased to 3%. After
    this time, the offspring showed statistically significant (P<0.05)
    body weight depressions on days 14 and 21; the males also had lower
    body weights than controls at day 28. The observed body weight
    depressions were slightly less than in the dose-response group from
    parents fed 5.0% sodium saccharin continuously but by day 28 the body
    weights of the male rats in these two groups were similar.

         The feeding of 5.0% sodium hippurate to Fo animals from weaning
    through a single reproductive cycle caused statistically significant
    (P<0.05) body weight depressions averaging up to 10% and 14% in male
    and female rats, respectively. In females, the average depression was
    only 4% until the beginning of gestation. Unlike the sodium saccharin
    treated animals, Fo rats fed sodium hippurate consumed less diet than
    controls. The feeding of sodium hippurate caused a statistically
    significant (P<0.05) decrease in the mean number of pups per litter
    at birth and aggressive behaviour and mortality in the lactating dams.
    Teratogenic effects (microphthalmia, domed heads and hydrocephaly) and
    statistically significant (P<0.05) depressions in body weight were
    noted in the offspring. The body weight depressions were as high as
    38% in 28 day-old males. Increased water intake and urine volume were
    observed in the Fo rats but the changes were less than in the
    corresponding 5% sodium saccharin group. Little or no visible changes
    in the faecal moisture content were observed.

    Results in the F1 generation

         There were a statistically significant trend for increased
    survival in the sodium saccharin treatment groups, being most evident
    at the 5.0% and 7.5% treatment levels. The survival rates after 123
    weeks of treatment were as shown:

    Treatment group                              Survival rate (%)
                                                                  

          Control                                        23
    1.0%  sodium saccharin                               24.5
    3.0%  sodium saccharin                               23
    4.0%  sodium saccharin                               19
    5.0%  sodium saccharin                               37
    6.25% sodium saccharin                               26
    7.5%  sodium saccharin                               34
    5.0%  sodium saccharin through gestation             20
    5.0%  sodium saccharin following gestation           36
    3.0%  sodium hippurate                               30

         No changes in behaviour or appearance were observed in the study.

         A clear dose-response was observed for physiological effects at
    treatment levels of 3.0% sodium saccharin or above. Changes such as
    relative depressions in body weight, food consumption and water
    consumption were seen but there was no direct statisticaly correlation
    with the occurrence of bladder tumours.  The 1.0% dietary
    concentration was considered a no-effect level for these changes.

         Urinanalysis revealed a dose-dependent increase in the mean 24 h
    urine volume and decrease in osmolality. These changes were primarily
    observed at dietary concentrations of sodium saccharin of >3.0% and
    showed a strong statistical correlation to the occurrence of bladder
    tumours. During the first 92 days, the urinary pH was significantly
    depressed for all groups in the dose-response portion of the bioassay
    in which sodium saccharin was fed at dietary concentrations >4%;
    significantly lower urinary pH values were also recorded in the 3%
    sodium saccharin group at the 64 and 92 day time intervals. No
    dose-related lowering of pH was evident later in the study (6-29 m)
    except for the 4% and 7.5% sodium saccharin groups at the 13 m time
    interval. At no stage in the study was the pH of the 1% sodium
    saccharin group significantly lower than control values. The urine pH
    values in the 5.0% sodium saccharin through gestation group were
    comparable to control values but significant depressions were noted in
    the 5% sodium saccharin following gestation group at the 6-, 30-, 64-
    and 92-day time intervals, and a statistically significant depression
    was also observed at 24 m for the 3% sodium hippurate group. No
    treatment-related changes were seen in the analyses for urinary
    urobilinogen, protein, glucose, ketones, bilirubin, occult blood and
    nitrite. Analysis of the urine for calcium ion was performed at the
    24 m interval and no treatment-related effects on calcium ion
    concentration were observed; however, due to the increased urine
    volume, there was a treatment-related increase in total calcium ion
    excreted (mg/24 h) in the groups receiving >3.0% sodium saccharin.
    Examination of the urine for microcrystals showed that at 13, 18, and
    24 m, the control group had significantly higher crystal scores than
    the 7.5% sodium saccharin group, and crystalluria did not appear to be
    involved in bladder tumour formation.

         At autopsy, there was a significant increase in absolute and
    relative bladder weights at sodium saccharin dietary concentrations of
    3% or above. No urinary bladder weight changes were noted in animals
    exposed to sodium saccharin only in utero nor in animals fed sodium
    hippurate.

         Histopathological examination revealed a treatment- and dose-
    related mineralization of the renal pelvis but no treatment-related of
    the ureter or urethra were observed.

         A clear dose response for bladder tumours was observed. The slope
    of the dose-response curve was steep indicating that the incidence of
    bladder tumours declined rapidly with decrease in dose. The incidence
    of bladder tumours observed in the various treatment groups was as
    follows:

    Treatment group          Incidence of primary bladder tumours %
                                  Benign    Malignant      Total
                                                                    

          Control                   0.0        0.0           0.0*
    1.0%  sodium saccharin          0.6        0.2           0.8
    3.0%  sodium saccharin          0.8        0.8           1.6
    4.0%  sodium saccharin          2.1        4.2           6.3
    5.0%  sodium saccharin          3.3        9.2          12.5
    6.25% sodium saccharin         10.0        6.7          16.7
    7.5%  sodium saccharin         15.3       16.1          31.4
                                                                    

    *    Tumour incidence in 863 control male rats of the same used in
         this study from 10 recent in utero lifetime studies conducted
         at IRDC ranged between 0.0-2.5% for papillomas and 0.0-0.8% for
         carcinomas. The mean incidence for total urinary bladder tumours
         was 0.8%.

         In this study, the lowest dosage level of 1.0% was considered a
    no-effect level for bladder tumours based upon pairwise statistical
    analyses with the concurrent untreated control group and a comparison
    with background bladder tumour incidence for this strain of rat at the
    IRDC laboratory utilizing an in utero lifetime design. At the 3.0%
    sodium saccharin treatment level, the incidence of benign bladder
    tumours alone or of malignant bladder tumours alone was not
    significantly increased, but the combined incidence was significantly
    higher than in concurrent controls. The incidence of benign and/or
    malignant bladder tumours was significantly increased at dosage levels
    of 4% or greater. No increase in the incidence of hyperplasia or other
    treatment-related effects were observed in the ureter, urethra or
    kidney.

         The animals exposed to saccharin only in utero were comparable
    to controls but the animals whose exposures began at birth (5% sodium
    saccharin following gestation) had an incidence of urinary bladder

    tumours similar to that of animals fed diets containing 5% sodium
    saccharin whose exposure included the in utero period. Therefore it
    appeared that in utero exposure was not essential to the development
    of urinary bladder tumours in sodium saccharin treated rats. No
    bladder tumours were seen in the group fed sodium hippurate although
    the incidence of kidney mineralization was similar to that in the 3.0%
    sodium saccharin group.

         No other treatment-related toxic or neoplastic lesions were
    observed in either the genito-urinary tissues or in the various
    tissues examined only in the event of a macroscopic lesion.

    Statistical considerations

         A detailed statistical analysis of the data from the IRDC
    dose-response carcinogenicity study was carried out by Carlborg
    (1983). The data were examined using four types of mathematical model
    for carcinogenic dose/response viz:

         the threshold level (no-effect-level) model,
         the one-bit model,
         the Weibull model, and
         the polynomial (multi-stage) model.

         Three versions of the polynomial model were considered; the first
    with a cubed power of the dose as the lowest term, the second with a
    squared power of the dose as the lowest term and the third with a
    linear power of the dose as the lowest term.

         A dose of 0.01% sodium saccharin in the diet was chosen as the
    level at which low-risk assessments were made.

         The data overwhelmingly rejected the one-hit model; all the other
    models fitted the data in the statistical sense. The threshold level
    method yielded 1.0% as a lower bound on the threshold . The
    linearized polynomial model yielded a best estimate of 5.9  10-5 for
    the excess risk at a saccharin dose of 0.01% of the diet with an upper
    confidence limit of 9.1  10-5. The Weibull model yielded a best
    estimate of 2.5  10-10 for the excess risk at a saccharin dose of
    0.0l% with an upper confidence limit of 1.2  10-8.

         The author concluded that, even under the conservative assumption
    of low-dose linearity, the results from the IRDC study have reduced
    the estimated risk by roughly one order of magnitude relative to the
    estimates based on previous experiments; a risk assessment based on
    the observable dose-response pattern showed that saccharin is
    virtually safe at an exposure of societal concern. Some of the
    measurable characteristics of the urine of the rat taken very early in
    life appeared to be predictive of tumourigenicity.

    Long-term studies

    Monkey

         In addition to the long-term rat study described above, Adamson &
    Sieber (1983) administered saccharin (25 mg/kg bw) orally to two
    groups of 10 monkeys each on 5 days/week. One group received saccharin
    for an average of 122 months and the second group for 36 months. Since
    the inception of the study, none of the animals have died and there is
    no evidence of toxicity or tumours in any of the animals.

    OBSERVATIONS IN MAN

    Epidemiological studies

         Morgan (1983) has reviewed the epidemiological studies carried
    out in relation to ingestion of saccharin by man, including new
    studies and re-analyses not previously available to JECFA (Walker et
    al., 1982; Hoover & Hartge, 1982; Jensen & Kamby, 1982; Morrison et
    al., 1982; Najem et al., 1982). In an attempt to summarize the studies
    done to date, a statistical power analysis of previous case-control
    studies was carried out. Based on this analysis, it was calculated
    that, if the true relative risk of bladder cancer as a result of using
    artificial sweeteners were 1.13 or larger, there was a 95% probability
    that the studies reviewed, in toto would have detected such a risk
    as statistically significant.

         The results reviewed demonstrated that saccharin is not a strong
    or even a moderate carcinogen for man and the author concluded that
    the remarkable approximation to unity of the summary relative risk
    from all studies was impressive.

    Comments

         New information presented to the Committee included biochemical, 
    pharmacokinetic, mutagenicity, and epidemiological data; the results
    of special studies on urine volume and composition and the effect of
    saccharin on the bladder epithelium; the results of studies on
    saccharin as a promoter or co-carcinogen; and the results of a
    carcinogenicity study in rats designed to investigate the dose-
    response relationship in the development of bladder tumours and the
    outcome of in utero exposure.

         In the Committee's opinion the available evidence indicated that
    saccharin is not mutagenic. An in utero phase of exposure is not
    essential for a carcinogenic response to saccharin in the bladder of
    the male rat. There was a definite carcinogenic effect at levels of
    dietary inclusion of 3% and above in the long-term study with in
    utero exposure. There was also a carcinogenic effect at a level of
    5%, the only level tested, in the 1-generation study with exposure

    from birth, which included pups suckled by dames receiving saccharin
    in their diets. The Committee considered that a 1% dietary inclusion
    level could be taken as a no-effect level. Further data on the bladder
    histopathology in the carcinogenicity study mentioned above were
    received too late to be reviewed by the Committee. Within the
    statistical limitations of the studies, the epidemiological data do
    not show any evidence that saccharin is a bladder carcinogen.

    EVALUATION

    Level causing no toxicological effect

         Rat: 1% (10.000 ppm) in the diet, equivalent to 500 mg/kg bw.

    Estimate of temporary acceptable daily intake for man

         0-2.5 mg/kg bw.

    FURTHER WORK OR INFORMATION

         (Information to be submitted when it becomes available.)

    1.   Data on the bladder histopathology.

    2.   Information to elucidate the mechanism by which the compounds
         produce bladder tumours, including the possible significance of
         exposure through lactation, the influence of gastrointestinal 
         tract microorganisms, the effect of osmolar changes in the urine,
         and species specificity in the development of urothelial changes.

    REFERENCES

    ADAMSON, R.H. & SIEBER, S.M. (1983) Chemical carcinogenesis studies in
    non-human primates. In: Organ and Species Specificity in Chemical
    Carcinogenesis. R. Langenbach et al., eds. p. 129, New York: Plenum.

    ANDERSON, R.L. (1979) Response of male rats to sodium saccharin
    ingestion: urine composition and mineral balance. Fd. Cosmet.
    Toxicol., 17; 195.

    ANDERSON, R.L. (1983) Effect of saccharin ingestion on stool
    composition in relation to caecal enlargement and increased stool
    hydration. Fd. Chem. Toxicol., 21: 255.

    BERNDT, W.O., REDDY, R.V., & HAYES, A.W. (1981) Evaluation of renal
    function in saccharin treated rats. Toxicology, 21: 305.

    CARLBORG, F.W. (1983) Statistical considerations in the design of the
    IRDC experiment with saccharin.  Unpublished report presented at the
    Scientific Review Conference, Duke University Medical Center, 4th-6th
    May, 1983.

    COLBURN, W.A., BEKERSKY, I., & BLUMENTHAL, H.P. (1981) A preliminary
    report on the pharmacokinetical of saccharin in man: single oral
    administration. J. Clin. Pharmacol. 21: 147.

    DEMERS, D.M., FUKUSHIMA, S., & COHEN, S.M. (1981) Effect of sodium
    saccharin and L-tryptophan on rat urine during bladder carcinogenesis.
    Cancer Res., 41: 108.

    EL-GERZAWI, S., HEATFIELD, B.M., & TRUMP, B.F. (1982) N-methyl-
    N-nitrosourea and saccharin: effects on epithelium of normal human
    urinary bladder in vitro. J. Natl. Cancer Inst., 69: 577.

    FAHRIG, R. (1982) Effects in the mammalian spot test: cyclamate versus
    saccharin. Mutation Res., 103: 43.

    FUKUSHIMA, S. & COHEN, S.M.  (1980)  Saccharin-induced hyperplasia of
    the rat urinary bladder. Cancer Res., 40: 734.

    FUKUSHIMA, S., HIROSE, M., OKUDA, M., NAKANOWATARI, J., HATANO, A.,
    & ITO, N. (1982) Effect of partial cystectomy on the induction of
    pre-neoplastic lesions in rat bladder initiated with N-butyl-N-
    (4-hydroxybutyl) nitrosamine followed by bladder carcinogens and
    promoters. Utol. Res., 10: 115.

    FUKUSHIMA, S., ARAI, M., NAKANOWATARI, J., HIBINO, T., OKUDA, M., &
    ITO, N. (1983a) Differences in susceptibility to sodium saccharin
    among various strains of rats and other animal species. Gann,
    74: 8.

    FUKUSHIMA, S., HAGIWARA, A., OGISO, T., SHIBATA, M., & ITO, N. (1983b)
    Promoting effects of various chemicals in rat urinary bladder 
    carcinogenesis initiated by N-nitroso-n-butyl-(4-hydroxybutil)
    amine. Fd. Chem. Toxicol., 21: 59.

    HOOVER, R.1N. & HARTGE, P. (1982) Non-nutritive sweeteners and bladder
    cancer. Am. J. Publ. Hlth., 72; 382.

    IMAIDA, K., OSHIMA, M., FUKUSHIMA, S., ITO, N., & HOTTA, K. (1983)
    Membrane potentials of urinary bladder epithelium in F344 rats treated
    with N-butyl-n-(4-hydroxybutil) nitrosamine or sodium saccharin.
    Carcinogenesis, 4: 659.

    IRDC (1983) Evaluation of the dose response and in utero exposure of
    saccharin in the rat. Unpublished report of the International Research
    & Development Corporation, Mattawan, MI.

    ISHII, D.N. (1982) Inhibition of iodinated nerve growth factor binding
    by the suspected tumour promoters, saccharin and cyclamate. J. Natl.
    Cancer Inst., 68: 299.

    ITO, N., FUKUSHIMA, S., SHIRAI, T., & NAKANISHI, K. (1983a) Effects 
    of promoters on N-butyl-N-(4-hydroxybutyl) nitrosamine-induced urinary
    bladder carcinogenesis in the rat. Environ. Hlth. Pers., 50: 61.

    JENSEN, O.M. (1983) Artificial sweeteners and bladder cancer:
    epidemiological evidence. Unpublished paper presented at the Third
    European Toxicology Forum, Geneva, Switzerland. October 18-22, 1983.

    LITTON BIONETICS INC. (1983a) Mutagenicity evaluation of FDC 12 in the
    Ames Salmonella/Microsome plate test. Unpublished Report submitted to
    FDC Consultants Inc.

    LITTON BIONETICS INC. (1983b) Mutagenicity evaluation of FDC 14 in the
    Ames Salmonella/Microsome plate test. Unpublished Report submitted to
    FDC Consultants Inc.

    LITTON BIONETICS INC. (1983c) Mutagenicity evaluation of Lot No.2517
    in the Ames Salmonella/Microsome plate test. Unpublished Report
    submitted to FDC Consultants Inc.

    LOK, E., IVERSON, F., & CLAYSON, D.B. (1982) The inhibition of urease
    and proteases by sodium saccharin. Cancer Letts., 16: 163.

    MAHON, G.A.T., & DAWSON, G.W.P. (1982) Saccharin and the induction of
    presumed somatic mutations in the mouse. Mutation Res., 103: 49.

    MATTHEWS, H.B., FIELDS, M., & FISHBEIN, L. (1973) Saccharin:
    distribution and excretion of a limited dose in the rat. J. Agr. Fd.
    Chem., 21: 916.

    MORGAN, R.W. (1983) A review of epidemiologic studies of artificial
    sweeteners and bladder cancer. Unpublished Report presented at the
    Scientific Review Conference, Duke University Medical Center, 4th-6th
    May, 1983.

    MORRISON, A.S., VERHOEK, W.G., LECK, I., AOKI, K., OHNO, Y., & OBATA,
    K. (1982) Artificial sweeteners and bladder cancer in Manchester,
    U.K., and Nagoya, Japan. Br. J. Cancer, 45: 332.

    MURASAKI, G. & COHEN, S.M. (1981) Effect of dose of sodium saccharin
    on the induction of rat urinary bladder proliferation. Cancer Res.,
    41: 942.

    MURASAKI, G. & COHEN, S.M. (1983a) Co-carcinogenicity of sodium
    saccharin and N-(4-(5-nitro-2-firyl)-2-thiazolyl) formamide for the
    urinary bladder. Carcinogenesis, 4: 97.

    MURASAKI, G. & COHEN, S.M. (1983b) Effect of sodium saccharin on
    urinary bladder epithelial regenerative hyperplasia following freeze
    ulceration. Cancer Res., 43: 182.

    NAJEM, G.R., LOURIA, D.B., SEEBODE, J.J., THIND, I.S., PRUSAKOWSKI,
    J.M., AMBROSE, R.B., & FERNICOLA, A.R. (1982) Life-time occupation,
    smoking, caffeine, saccharine, hair dyes and bladder carcinogenesis.
    Int. J. Epid., 22: 212.

    NAKANISHI, K., FUKUSHIMA, S., HAGIWARA, A., TAMANO, S., & ITO, N.
    (1982) Organ specific promoting effects of phenobarbital sodium and
    sodium saccharin in the induction of liver and urinary bladder tumors
    in male F344 rats. J. Natl. Cancer Inst., 68: 497.

    PANTAROTTO, C., SALMONA, M., & GARATTINI, S. (1981a) Plasma kinetics
    and urinary elimination of saccharin in man. Toxicol. Lett.,
    9: 367.

    PANTAROTTO, C., SALMONA, M., FANELLI, R., BIACHI, M., & SZCZAWINSKA,
    K.  (1981b) ' GLC-mass fragmentographic determination of saccharin in
    biological fluids. J. Pharmaceut. Sci., 70: 871.

    RADFORD, T., COOK, J.M., DALSIS, D.E., WOLF, E., & VOIGT, M. (1983)
    Identification and quantitation of aminosaccharins in sodium saccharin
    produced by the Maumee process. Fd. Chem. Toxicol., in press.

    REITZ, R.H., FOX, T.R., QUAST, J.F., EMERSON, J., LEWKOWSKI, J., &
    STANLEY, J. (1983) Food consumption patterns and effects of saccharin
    upon urinary bladder tissue in very young rats. Unpublished report,
    Dow Chemical, U.S.A.

    REITZ, R.H. & MENDRALA, A.L. (1983) Evaluation of soldium saccharin
    and 1-naphthalene sulfonic acid in the rat hepatocyte unscheduled DNA
    synthesis assay. Unpublished report, Dow Chemical, U.S.A.

    RENWICK, A.G. & SIMS, J. (1983) Distension of the urinary bladder in
    rats fed saccharin containing diet. Cancer Letts., 18: 63.

    RIGGIN, R.M. & KINZER, G.W. (1983) Characterization of impurities in
    commercial lots of sodium saccharin produced by the Sherwin-Williams
    process 1. Chemistry. Fd. Chem. Toxicol., 21: 1.

    SCHOENIG, G.P. & ANDERSON, R.L. (1983) The effect of high dietary
    levels of sodium saccharin on selected physiological parameters in
    rats. Unpublished report.

    SHIBATA, S., GOLDSTEIN, I.J., & KIRKLAND, J.J. (1983) Structure of a
    water-insoluble D-glucan isolated from a Streptococcal organism.
    Carbohydrate res., 120: 77.

    SIMS, J. & RENWICK, A.G. (1983a) Unpublished observations cited by
    Renwick, A.G. (1983) Saccharin and tryptophan metabolism. Paper
    presented at the Expert Review Panel Meeting, Duke University, 4th-6th
    May, 1983.

    SWEATMAN, T.W. & RENWICK, A.G. (1980) The tissue distribution and
    pharmaeokinetics of saccharin in the rat. Toxicol. appl. Pharmacol.,
    55: 18.

    SWEATMAN, T.W., RENWICK; A.G., & BURGERS, C.D. (1981) The
    pharmacokinetics of saccharin in man. Xenobiotica, 11: 531.

    SWEATMAN, T.W. & RENWICK, A.G. (1982) Tissue levels of saccharin in
    the rat during two-generation feeding studies. Toxicol. appl.
    Pharmacol., 62: 465.

    TSING, M.T. (1983) Final report on the preliminary autoradiographic
    evaluation of the urinary bladder from saccharin treated rats.
    Unpublished report.

    TSUDA, H., FUKUSHIMA, S., IMAIDA, K., KURATA, Y., & ITO, N. (1983)
    Organ-specific promoting effect of Phenobarbital and Saccharin in
    induction of thyroid, liver and urinary bladder tumours in rats after
    initiation with N-nitrosomethylurea. Cancer Res., 43: 3292.

    WEST, R.W. & JACKSON, D.C. (1981) Saccharin effects on the urinary
    physiology and urothelium of the rat when administered in diet or
    drinking water. Toxicol. Lett., 7: 409.

    WALKER, A.M., DREYER, N.A., FRIEDLANDER, E., Loughlin, J., ROTHMAN,
    K.J., & KOHN, H.I. (1982) An independent analysis of the National
    Cancer Institute Study on non-nutritive sweeteners and bladder cancer.
    Am. J. Pub. Hlth., 72: 376.

    WESTLAND, J.A. & HELTON, E.D. (1983) Evaluation of sodium saccharin
    administered in dry and liquid diets in Rhesus monkeys. Unpublished
    report of the Primate Research Institute, New Mexico State University.

    WOLF, E. & VOIGT, M. (1983) Determination of polar impurities in
    saccharin and saccharin sodium by HPLC. Fresenius Z. Anal. Chem.,
    315: 135.
    


    See Also:
       Toxicological Abbreviations