Saccharin was evaluated by the Joint Expert Committee on Food
    Additives in 1967, 1974, 1978 and 1980 (see Annex I, Refs. 14, 34,
    48 and 54). In 1978, 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 animal studies which indicated that excessive and long-term
    ingestion of saccharin was potentially a carcinogenic hazard for
    humans. At the 1980 meeting the temporary ADI of 2.5 mg/kg was
    extended pending the completion of current investigations. Further
    studies have been received and evaluated and the previous monograph
    has been revised.

         Saccharin may be produced in various ways, starting from toluene
    (Remsen & Fahlberg, 1879), phthalic anhydride or phthalic acid
    (Maumee, 1951) and o-chlorotoluene (Bungard, 1967). Accordingly, a
    series of different chemical impurities could find their way to the
    final product. The most widely used methods for the manufacture of
    saccharin are the Remsen-Fahlberg and the Maumee processes (Munro et
    al., 1974).

         Although a universally applicable method for identification of
    impurities in saccharin regardless of its production method is not
    available, a recently published procedure (Stavric et al., 1976) for
    isolation and identification of organic solvent soluble impurities
    gives very satisfactory results for saccharin produced by the Remsen-
    Fahlberg method. The major impurity in saccharins produced by this
    procedure was identified as o-toluene-sulfonamide, one of the
    intermediate reaction products in the preparation of saccharin
    (Stavric et al., 1974b). Methods for isolation, identification and
    quantitation of water-soluble impurities have also been reported
    (Stavric et al., 1974a; Nelson, 1976).



         Saccharin and saccharin salts (sodium, ammonium, calcium) have
    been in use since the late nineteenth century, salt forms being more
    soluble but of the same sweetening power as the acid form.


         The absorption of ingested saccharin in animals and man occurs
    rapidly. With a pKa of 2.2, saccharin exists in acidic media
    predominantly in the unionized form, which is the more readily
    absorbed form in a number of animal species. Saccharin is more
    completely absorbed from the guinea-pig (pH 1.4) and rabbit (pH 1.9)
    stomach, than from the rat's stomach (gastric pH 4.2) (Ball, 1973;
    Minegishi et al., 1972). In vitro perfusion of rat stomach and small
    intestine with a solution of saccharin demonstrated considerable
    absorption from the stomach at pH 1.0 and slow absorption from the
    small intestine, i.e. less than 9% in 2 hours (Kojima et al., 1966).
    The gastrointestinal absorption of saccharin appears to be somewhat
    greater in monkeys than in rats (Pitkin et al., 1971a). In monkeys,
    and also most probably in man, both gastric acidity and degree of
    absorption are intermediate between those of the rabbit and guinea-pig
    on one side, and the rat on the other (Ball, 1973). This also means
    that the degree of absorption of saccharin could be dependent on food
    intake which affects the acidity of the gastric contents.

    Distribution and excretion

         Lethco & Wallace (1975) studied the distribution of radioactivity
    in organs and tissues of rats at various time intervals (1, 2, 4, 8,
    24, 48 and 72 hours) following a single oral administration of
    (3-14C)saccharin (50 mg/kg). Traces of radioactivity were found in
    almost all organs 1 hour after dosing. Fat, brain and spleen contained
    only minute quantities of 14C. Kidney, urinary bladder and liver
    contained the highest amount of 14C, which peaked at 4 and 8 hours.
    In subsequent experiments, rinsing the bladders of the treated rats
    with 8, 0.5 ml portions of a 0.9% saline solution, they found that a
    significant portion of the 14C activity was retained by or bound to
    the bladder tissue (Lethco & Wallace, 1975).

         In similar experiments, Matthews et al. (1973) found that a
    single oral dose (1 mg/kg bw) of 14C-saccharin (labelled in the
    carbonyl group) was rapidly absorbed from the gastrointestinal tract,
    with peak tissue radioactivity occurring within 15 minutes of dosing.
    With repeated dosing, within a single day or over a period of several
    days, there was an accumulation of saccharin in some tissues,
    particularly in the urinary bladder. The bladders of rats which
    received daily doses of saccharin contained 19 times as much saccharin
    as did the bladders of animals which received only a single dose.
    After the removal of saccharin from the diet, almost complete tissue
    clearance resulted in 3 days (Matthews et al., 1973).

         Saccharin crosses the placenta of the rat (West, 1979; Ball et
    al., 1977) and monkeys (Pitkin et al., 1971a,b) to a limited extent.
    However, clearance from foetal tissues may be slower than from
    maternal tissues, particularly the urinary bladder (Ball et al., 1977;
    Pitkin et al., 1971a,b). Additionally, saccharin has also been found
    to be excreted in the milk of goats (Carlson et al., 1923) and rats
    (West, 1979).

         Recently, Sweatman & Renwick (1980) studied the tissue
    distribution and pharmacokinetics of saccharin in a series of
    experiments. When saccharin was included in the diet of rats at a
    level of 5%, there was a marked diurnal variation in the concentration
    of saccharin in their plasma. Feeding male rats diets containing 1-10%
    saccharin for 22 days resulted in tissue concentrations of saccharin
    in the kidney and bladder which were greater than the concentration of
    saccharin in the plasma, while the concentrations of saccharin in the
    liver, lungs, spleen, adrenal, fat and muscle were less than in the
    plasma. When female rats were fed a diet containing 5% saccharin, the
    concentration of saccharin in the plasma and organs was greater than
    for the male rats fed the 5% diet, particularly the concentration in
    the kidney and urinary bladder. Additionally, the concentrations of
    saccharin in the plasma and tissues of the male rats fed the diets
    containing 7.5% or 10% saccharin had a higher than predicted
    concentration, based on a linear extrapolation from the lower dose
    levels. The authors attributed this to the animals' reduced ability to
    eliminate saccharin.

         A single i.v. dose of [3H] saccharin was given to male and
    female anaesthetized rats at levels from 1 to 1000 mg/kg. The plasma
    clearance of the [3H] saccharin was found to be dose dependent with
    the high dosages resulting in a 60% decrease in clearance. When rats
    were pretreated with probenecid prior to the i.v. administration of
    [3H] saccharin at levels of 50-200 mg/kg, a 70% reduction in plasma
    clearance was observed (Sweatman & Renwick, 1980). These findings were
    similar to those of Bourgoignie et al. (1980) who used unanaesthetized
    male and female rats to examine the mechanism by which saccharin was
    excreted via the kidneys, using a range of [3H] saccharin
    concentrations from 1 to 80 mg/dl in plasma. The authors concluded
    that saccharin was excreted by a combination of filtration and tubular
    excretion without resorption. The latter appeared to be carrier
    mediated, since competitive inhibition was observed between saccharin
    and para-aminohippurate.


         During its long history of use, saccharin has been accused of
    being responsible for a number of adverse effects both in human
    beings and laboratory animals. Saccharin has been implicated in the
    development of photosensitive skin eruptions in humans (Taub, 1972),
    and as a hypoglycaemic agent in animals (Thompson & Mayer, 1959). Low

    dietary concentrations of saccharin have been shown to alter the
    concentration of serum lipid components in rats fed chemically-defined
    diets (Purdom et al., 1973).

         No effect was noted on human nitrogen balance or protein
    utilization in man by doses up to 4 g of saccharin/day. Daily doses of
    5 g of saccharin reduced albumin absorption and utilization by 0.94%
    (Neumann, 1926a,b). No abnormal effects on total urinary nitrogen
    excretion or uric acid output were noted by other investigators after
    giving saccharin orally (Folin & Herter, 1912). No deleterious effects
    on blood sugar, kidney function, vitamin utilization, blood
    coagulation or enzyme activity were detected in man (NAS-NRC, 1955).


         It has long been assumed that saccharin undergoes very little
    metabolic conversion under normal dietary usage in animals and man
    (NRC, 1974).


         Four male and 2 female Sprague-Dawley rats were dosed with
    14C-saccharin (40 mg/kg) uniformly labelled in the benzene ring. More
    than 90% of the radioactivity was recovered in the urine collected for
    96 hours after dosing. Only 1 radioactive spot was detected on the TLC
    of the whole urine, which was identified as saccharin (Byard &
    Golberg, 1973). Three to 4% of the dose was excreted in the 48-hour
    collection of faeces as unmetabolized saccharin. During the first
    4-8-hour collection period, no more than 0.3% of an oral dose of
    14C-saccharin was excreted in the bile as unchanged saccharin.
    Induction of mixed function oxidase activity in rat liver by treatment
    with phenobarbitone had no influence on saccharin metabolism (Byard &
    Golberg, 1973).

         Kennedy et al. (1972), using albino rats treated with 10 or 20 µC
    of sodium saccharin labelled with 14C in the carbonyl group, obtained
    rapid excretion with total recovery from 90.21% to 100.57%. In 3 of
    the 4 rats the amount recovered in the urine ranged from 82.65% to
    96.95%, while in 1 animal it was only 67.91%. However, in this animal
    a total of 31.34% of the radioactive material appeared in the faeces.
    Most of the radioactivity excreted was within 24 hours of ingestion.
    Expired air and tissues contributed very little to the total 14C
    recovery. The same authors demonstrated the presence of trace amounts
    of 2 hydrolytic products of saccharin (o-sulfamoylbenzoic acid and
    ammonium carboxybenzene sulfonate) in urinary extracts. Minegishi et
    al. (1972) failed to demonstrate the presence of any hydrolytic
    products in the urine of rats given 35S-saccharin. Urine contained
    70% of the administered saccharin and the remainder was in the faeces.
    Similar results were found for rats given repeated daily doses of
    saccharin for 4-5 weeks prior to 35S-saccharin administration,

    indicating the lack of an induction mechanism for saccharin metabolism
    (Minegishi et al., 1972). Lethco & Wallace (1975) obtained 56-87% of
    the administered 14C-carbonyl labelled saccharin in urine and 10-40%
    in the faeces. Ninety-nine per cent. of the urinary 14C was unchanged
    saccharin, while the remainder was identified as the metabolite
    o-sulfamoylbenzoic acid. Matthews et al. (1973) also failed to
    demonstrate the presence of any saccharin metabolites in the urine of
    rats given multiple low doses of 14C-saccharin (1 mg/kg bw).

    Mouse, hamster, rabbit, guinea-pig and dog

         Metabolism and rates of saccharin elimination appear similar in
    these species. The fate of radioactive saccharin in the rabbit was
    studied by Ball (1973) and by Lethco & Wallace (1975). Lethco &
    Wallace (1975) made comparative studies of the metabolism of saccharin
    in dog, rabbit, guinea-pig and hamster. Similar comparative studies by
    Byard (1972) were done with mice, golden hamsters, guinea-pigs and
    dogs, while Minegishi et al. (1972) studied saccharin metabolism in
    guinea-pigs. These authors concluded that these species do not
    metabolize saccharin.

         Ball et al. (1974) failed to detect any metabolites in the urine
    of female Wistar albino rabbits treated orally with a single dose of
    20 mg/kg of (3-14C)saccharin. Seventy-two per cent. of the 14C was
    excreted in the urine in 24 hours and 15% in the faeces. In 48 hours,
    96% of the radioactivity was recovered. Similarly no metabolites were
    found in urine of rats treated in the same way, which were kept up to
    12 months on diets containing 1% or 5% sodium saccharin.


         Following a single oral dose of uniformly ring-labelled
    14C-saccharin (1 mg/kg and 10 mg/kg) to young female rhesus monkeys,
    98% of the radioactivity was excreted in the urine within 96 hours.
    A very small amount of 2 hydrolytic products of saccharin
    (o-sulfamoylbenzoic acid and ammonium carboxybenzene sulfonate) were
    detected in urinary extracts (prepared by ethyl acetate extraction of
    acidified urines) (Pitkin et al., 1971a). Byard & Golberg (1973) found
    that 14C-saccharin (40 mg/kg) was not metabolized in male rhesus
    monkeys. They also demonstrated that "artifactual metabolites" of
    saccharin were produced if the urine was extracted under acidic
    conditions. No induction of saccharin metabolism was found in groups
    of rhesus monkeys receiving sodium saccharin (0, 20, 100 and
    500 mg/kg/day) for 79 months. Metabolic studies conducted using
    radioactive saccharin with these monkeys on several occasions during
    the test indicated rapid, almost exclusively urinary excretion of
    unmetabolized saccharin (McChesney et al., 1977).


         Orally administered saccharin appeared in the urine of man within
    a half hour of dosing and was completely eliminated unchanged in 16-18
    hours (Staub & Staehelin, 1936), some 90% being excreted in the urine
    within 24 hours (Folin & Herter, 1912). Intravenous sodium saccharin
    in doses of 2.5 g has been used without adverse effects in sick and
    healthy people to determine circulation time (Fishberg et al., 1933).
    Studies conducted with human subjects (McChesney & Golberg, 1973)
    suggested that some degree of saccharin metabolism may occur in man,
    since oral doses of saccharin could not be recovered quantitatively as
    saccharin in the urine. Subsequent, more definitive studies, using
    14C-labelled saccharin (Byard et al., 1974) demonstrated that the
    reduced recovery was an artifact thought to be due to the binding of a
    portion of the urinary saccharin to unidentified urinary constituents.

         Four men received 500 mg of 14C-saccharin (uniformly labelled in
    the benzene ring) and excreta was collected at intervals up to 96
    hours post-dosing. More than 98% of the 14C was recovered within 48
    hours (92.3% in urine, 5.8% in faeces). An additional 0.3% was
    excreted in the 48-72-hour collection interval. None of the detected
    saccharin was found to have been metabolized. Consequently, it was
    concluded that man, like other species, does not metabolize saccharin
    (Byard et al., 1974).


    Special studies on carcinogenicity: other than dietary exposure

         Saccharin and croton oil together were tested for dermal
    co-carcinogenicity in mice. Although the treated group showed a
    greater incidence of skin papillomas compared with the control, the
    difference was not statistically significant (Salaman & Roe, 1956).

         Paraffin wax pellets containing saccharin when implanted in the
    mouse bladder induce a significant incidence of bladder tumours and
    this was interpreted as demonstrating a co-carcinogenic effect (Allen
    et al., 1957).

         Hicks et al. (1973, 1975) reported a rat model system for testing
    bladder carcinogens wherein each rat was subjected to a pre-test
    treatment consisting of a single instillation into the bladder of up
    to 2 mg of methyl-N-nitrosourea (MNU). The test treatment consisted of
    adding saccharin either to the diet or drinking-water at dosage levels
    up to 4 g/kg/day. This treatment produced a significantly higher
    incidence of bladder tumours with a dramatically reduced latent period
    in the MNU plus saccharin treated rats compared to the untreated
    control or animals receiving a single instillation of MNU or animals
    receiving saccharin in their diet or drinking-water without any MNU
    pretreatment. Although there does not appear to be any correlation

    between the incidence of bladder tumours or bladder calculi (Hicks et
    al., 1975), considerably more research is needed to evaluate the
    biological significance of this model. Additionally, other
    laboratories have been unable to reproduce these results (Mohr et al.,
    1978; Green & Rippel, 1979; Green et al., 1980; Hooson et al., 1980).
    Some of the suggested experimental variables which may have affected
    the results include the quantity of MNU required to produce a
    subcarcinogenic or initiator dose (Hooson et al., 1980); the length
    and/or storage conditions for MNU; purity of MNU; or time lapse
    between preparation of the MNU solutions and the treatment of the
    animals (Mohr et al., 1978).

         Subsequently, Cohen et al. (1978, 1979) developed a similar
    2-stage cancer model wherein N[4-(5-nitro-2-furyl)-2-thiazolyl]
    formamide (FANFT) served as the initiator, being administered in the
    diet to rats at a level of 0.2%. In their initial studies, FANFT was
    included in the diet for 6 weeks prior to feeding a saccharin (5%)
    containing diet or a control diet for 2 weeks prior to the saccharin
    (5%) diet. Both regimes resulted in a significant increase in the
    incidence of bladder tumours. In subsequent studies on the rat, the
    use of FANFT was decreased to 4 (Fukushima et al., 1981) and then 2
    weeks (Cohen et al., 1982). The latter study employed various
    treatment regimes that included FANFT- and saccharin-containing diets,
    freeze ulceration, and the i.p. injection of cyclophosphamide. The
    authors found that when bladder mucosal proliferation was increased,
    following freeze ulceration and cyclophosphamide injection, the
    bladder appears to be more sensitive to the promoting effects of

         Using an experimental model similar to that of Cohen et al.
    (1979), Nakanishi et al. (1980) administered N-butyl-N-
    (4-hydroxybutyl) nitrosamine (BBN) in the drinking-water at a level of
    0.001% for 4 weeks, either concurrently with or prior to feeding a
    diet containing 5% saccharin. In the co-administration study,
    saccharin enhanced the induction of urinary bladder hyperplasia
    (simple, papillary or nodular) and papillomas, while the sequential
    administration only enhanced the induction of papillary or nodular

         In vitro promotional assay systems using saccharin have also
    been reported. Mondal et al. (1978) reported that an initiating but
    non-transforming dose of 3-methylcholanthrene (3MC) was needed in a
    C3H/10T1/2 oncogenic transformation system before saccharin produced a
    significant number of transformed cells. Sivak & Tu (1980) initiated
    BALB/c3T3 cells with 3MC prior to the addition of saccharin, which was
    not found to be active as a direct transforming or promoting agent of
    Type III transformed foci.

    Special studies on carcinogenicity: feeding studies


         Groups of 50 female Swiss mice (9-14 weeks of age) were fed one
    of the following diets for 18 months: control (2 groups); 10% sucrose;
    or 5% saccharin. Seven days prior to the start of the study, half of
    the mice received a single intragastric instillation of 0.2 ml
    polyethylene glycol while a similar group of mice received an equal
    amount of polyethylene glycol which contained 50 µg benzo(a) pyrene
    (BP), to ascertain whether the dietary treatments might be
    co-carcinogenic with BP. Neither body weight nor survival in any of
    the treatment groups was different from controls. The administration
    of BP clearly increased the incidence of neoplasms arising in the
    forestomach epithelium, but the other dietary treatments had no effect
    upon this type of neoplasm. Although no neoplasms of the urinary
    bladder were seen on careful macroscopic examination of all animals,
    the bladders were not examined microscopically (Roe et al., 1970).

         Kroes et al. (1977) fed diets containing 0, 0.2 and 0.5% sodium
    saccharin during a 6-generation study in which 50 male and 50 female
    mice from the F0, F3b and F6a generations remained on the test diets
    for 20 months. There were no significant treatment-related tumorigenic

         In a study performed in duplicate at the Bio-Research Consultants
    Inc., groups of 25 male and 25 female mice (8 weeks of age) were fed
    diets containing 0, 10 000 or 50 000 ppm (0, 1 or 5%) saccharin in the
    diet for 24 months. The incidence of bladder tumours in the various
    groups was as follows: male controls, 1 of 19; 1% saccharin (males),
    none of 14 in the first replicate and none of 15 in the second; 5%
    saccharin (males), 1 of 15 in the first replicate and 2 of 19 in the
    second. Similar groups of female mice were also tested and none of the
    female mice developed bladder tumours (Homburger, 1978).

         Miyaji (1974) fed 50 male and 50 female mice (30 days of age)
    diets containing 0, 0.2, 1.0 or 5.0% saccharin for 21 months. No
    adverse treatment-related effects were reported.


         Groups of 30 male and 30 female hamsters, 8 weeks of age,
    received the following levels of saccharin in their drinking-water for
    the balance of their lifetime: 0, 0.156, 0.312, 0.625 or 1.25%.
    Average survival time was 50-60 weeks. The overall incidence of
    tumours in the control animals was 10.1% in 168 controls, while in the
    saccharin group consisting of 299 animals the incidence was 14.7%. The
    tumour types were similar in both groups and no urinary tract
    neoplasms were found in either group (Althoff et al., 1975).

    Rat (1-generation feeding studies)

         Fitzhugh et al. (1951) performed the first 2-year saccharin
    feeding study, where rats received a control diet (7 male and 9
    female), or diets containing 1.0% (10 male and 10 female) or 5.0%
    saccharin (9 male and 9 female). Saccharin had no apparent effect upon
    mortality, haematology or organ weight (liver, kidney and spleen), but
    did result in a slight growth depression in the 5% group. The only
    significant pathological changes observed were in the 5% group where 7
    animals (sex unspecified) had lymphosarcomas as well. The urinary
    bladder was not histologically examined.

         In another study, groups of 20 male and 20 female rats were fed
    diets containing 0, 0.005, 0.05, 0.5 or 5.0% saccharin for 2 years. A
    similar group was also given 1 ml of a 1% aqueous solution of trypan
    blue s.c. every 2 weeks for 1 year as a positive control. In the 5%
    group and the trypan blue group, mortality was higher than in the
    controls. Mortality was lower than for the controls in the 0.005%
    group. Retardation of growth was observed in the males and females of
    the 5% group despite greater feed consumption.

         One female and 4 male rats in the 5% saccharin group had bladder
    calculi and 1 male had kidney calculi. The female rat with calculi had
    an extensive transitional cell papilloma of the bladder, while another
    5% female without calculi had hyperplasia and papillomatosis. No
    evidence of nematodes was found in any of these animals (Lessel,

         Schmahl (1973) reported spontaneous tumours in rats which were
    fed diets containing either 0, 100 or 250 mg/kg of sodium saccharin
    (52 males and 52 females per group). The incidence of Trichosomoides
    crassicauda was 16% and was associated with a mild cystitis
    condition, but no bladder tumours were observed in the control or
    saccharin-treated groups.

         Miyaji (1974) fed groups of 54 male rats, which were 40 days of
    age, diets containing 0, 0.2, 1.0 or 5% saccharin for 28 months. No
    significant tumorigenic effects were observed.

         In a study conducted by Litton Bionetics, which was performed in
    duplicate, 26 male and 26 female rats were fed diets containing 0, 1
    or 5% saccharin for 24 months (NRC, 1974). The following was reported:
    "The incidence of all tumors at 24 months in the untreated control
    groups was 45% and 60% for the males, 80% and 55% for the females in
    the two replicate experiments. It is important to note the extent of
    the variation, indicative of the great variability of the assay as
    well as the high tumor incidence in the controls."

         However, "a single papilloma of the urinary bladder was found in
    a female of the high dose saccharin group in the second replicate; no
    other bladder tumor was found in the study".

         Homburger (1978) fed groups of 25 male rats (8 weeks of age)
    diets containing 0, 10 000 or 50 000 ppm (0, 1 or 5%) saccharin for 24
    months. The study was performed in duplicate. The incidence of bladder
    tumours in the various groups was as follows: controls, 1 of 16; 1%
    saccharin, 1 of 13 in the first replicate and 1 of 15 in the second;
    5% saccharin, 1 of 12 in the first replicate and none of 14 in the

         Groups of 54-56 male rats were fed either a control diet or a
    diet containing 2.5 g of sodium saccharin/kg/day for 28 months. Ten to
    16 rats in each group were killed at 12 months. Haematological-blood-
    biochemistry and pathological examinations were performed on the rats
    when sacrificed. The internal organs of all rats dying during the
    study were examined macroscopically. A significant growth depression
    was observed in the treated group, but no differences in mortality
    were found. No bladder abnormalities were reported for either control
    or treated animals (Furuya et al., 1975).

         Groups of 60 male and 60 female weanling rats were fed diets
    containing sodium saccharin produced by the Remsen-Fahlberg procedure
    to provide daily doses of 0, 90, 270, 810 or 2430 mg of saccharin/kg/
    day. The study ran for 26 months. Food consumption, body weight and
    clinical examinations were conducted weekly on all rats. The animals
    were free of the bladder parasite Trichosomoides crassicauda. One
    bladder tumour was found in a male control, 1 each in a male and
    female of the 90 mg/kg group and 2 male animals in the 810 mg/kg
    group. The tumours were all transitional cell papillomas. Three
    bladder calculi were observed grossly and 67 animals were found to
    have bladder calculi small enough to pass through the urethra. The
    presence of bladder calculi was not associated with the saccharin
    treatment or with the presence of bladder tumours. Saccharin
    administration was not accompanied by an increase in tumour incidence,
    although high doses were associated with reduced body weight in both
    sexes. No decrease in food consumption was observed. There was a
    decreased longevity in male rats. Diarrhoea was observed in the
    highest dose group, but was not accompanied by pathological evidence
    of enteritis. The haematological parameters measured were not affected
    by saccharin and saccharin had no effect upon the chemical composition
    of the urine (Munro et al., 1975).

    Rat (2-generation studies)

         Tisdel et al. (1974) fed 20 male and 20 female rats one of the
    following saccharin-containing diets for approximately 3 months prior
    to mating: 0, 0.05, 0.5 or 5.0%. The saccharin used in this study was
    produced by the Remsen-Fahlberg procedure. Following weaning, the F0
    generation was killed and the F1 generation was weaned on to their
    parents' diet, which they consumed until the study was terminated 100
    weeks later. Thus, the F1 generation animals were exposed to
    saccharin and its impurities during gestation, throughout lactation

    via the dam's milk and for the remainder of their lifetime via the
    diet. Transitional cell carcinomas of the urinary bladder were found
    exclusively in the males of the 5.0% group with an incidence rate of 7
    animals. One male in the 0.5% group had an epithelial hyperplasia of
    the urinary bladder which the authors considered to be of a
    precancerous type. Although no bladder tumours were observed in the
    female rats, squamous cell carcinomas of the uterus were found in 1
    animal in the 0.05% group and 2 animals in the 0.5% and 5.0% groups.

         In a combination 3-generation chronic feeding study reported by
    Taylor & Friedman (1974) and Taylor et al. (1980), 10 males and 20
    females were fed one of the following diets containing sodium
    saccharin produced by the Remsen-Fahlberg procedure: 0, 0.01, 0.1,
    1.0, 5.0 or 7.5%. These animals were mated after 3 months on test on a
    1 male to 2 female basis. The pups were weaned on to their parents'
    diet and the F0 generation was killed. The study was terminated after
    28 months. The reproductive data for this study are presented in
    another section. The average body weights of treated groups was
    generally less than that of the control animals; the lowest body
    weights were observed in the 7.5% group in which the body weight was
    depressed by 15% compared to the control, but survival was not
    affected by dietary treatment. The most significant pathological
    finding was the incidence of bladder tumours, with 1 tumour being
    found in a male control, 1 in a male on 5% sodium saccharin and a
    total of 9 fed the 7.5% sodium saccharin diet (7 males and 2 females).
    All the tumours were diagnosed as transitional cell carcinomas except
    for 1 tumour in a male in the 7.5% saccharin group which was diagnosed
    as a transitional cell papilloma.

         Since the Tisdel et al. (1974) study and the study by Taylor &
    Friedman (1974) have used saccharin which contained levels of o-TS up
    to 4660 ppm (0.466%) (Stavric et al., 1973), it was suggested (NRC,
    1974) that o-TS might be the chemical responsible for the bladder
    tumours observed in these 2-generation studies.

         Arnold et al. (1977, 1980) fed groups of 50 male and 50 female
    rats one of the following diets for 141 weeks: control, 2.5 mg
    o-TS/kg/day, 25 mg o-TS/kg/day, 250 mg o-TS/kg/day with 1% NH4Cl in
    the drinking-water or 5% sodium saccharin. The saccharin used in these
    experiments was produced by the Maumee procedure containing no
    detectable amounts of o-TS. After the F0 animals had been on test for
    3 months, they were mated on a 1:1 basis. The pups were weaned on to
    their parents' diet and both generations remained on their respective
    diets for their lifetime (30-32 months). The reproductive data are
    presented in another section. The animals were free of the bladder
    parasite Trichosomoides crassicauda. The only treatment-related
    effects, other than on lactation index and litter size, were a
    decreased growth rate in the 2 250 mg/kg o-TS groups and the saccharin
    group, and the incidence of bladder tumours.

         This is the first study where F0 male animals receiving sodium
    saccharin in their diet had a statistically higher incidence of
    bladder tumours than did the control groups (P <0.05). The authors
    suggest this observation may be attributable to the length of time on
    test plus the fact that the animals were only 30 days old when the
    experiment was initiated.

    Special epidemiological studies

         Available reports of epidemiological studies do not provide clear
    evidence to support or refute an association between bladder cancer in
    males and the use of saccharin.

         Evaluation of these studies on saccharin is difficult for many
    reasons. In some cases, only a very limited number of patients was
    studied. In others, a distinction between consumption of saccharin and
    cyclamates was not made, or smoking had a confounding effect.

         In 2 recently published epidemiological studies in humans, the
    authors concluded that no association between saccharin consumption
    and the incidence of bladder cancer was present (Morrison & Buring,
    1980; Wynder & Stillman, 1980).

    Special studies on mutagenicity

         A review on this subject was recently published (Kramers, 1975).
    A predominant feature emerging from a review of the literature on
    saccharin's mutagenic effects is a lack of consistency in the results.
    As an explanation, it was suggested that the detected effects might be
    due to a contaminant(s), present in varying amounts in different
    samples of saccharin (Kramers, 1975).

         This suggestion is supported by the findings of Stoltz et al.
    (1977) who reported mutagenic activity with the Ames Salmonella assay
    for some, but not all, samples of organic solvent extracts of various
    saccharin samples produced by the Maumee and Remsen-Fahlberg methods.

         The US Technology Assessment panel (OTA, 1977) commissioned a
    battery of 12 short-term tests, of which 10 had been completed prior
    to the publication of the panel's report. The sister chromatid
    exchange studies conducted with human lymphocytes, the mouse lymphoma
    forward mutation test and the Chinese hamster ovary cell test for
    chromosome aberration all gave positive results, while negative tests
    included the Ames Salmonella test, mitotic recombination in yeast,
    unscheduled DNA synthesis, the Pol A test, Drosophila sex-linked
    recessive lethal test, in vitro transformation test and the
    induction of plasminogen activator. Subsequently, Mondal et al. (1978)
    reported that saccharin did not produce oncogenic transformation of
    mouse embryo fibroblasts (C3H/10T1/2) unless an initiating dose of
    3-methylcholanthrene was used.

         Batzinger et al. (1977) orally administered saccharins of
    differing degrees of purity to mice and then collected the urine to
    test for mutagenic activity in the Ames Salmonella test. By modifying
    the growth and selection medium, they were able to detect mutagenic
    activity for all but the most purified samples of saccharin.

         When yeast was grown in a medium containing saccharin of
    different purities, Moore & Schmick (1979) found an inverse
    relationship between the incidence of mitotic crossing-over and the
    purity of the saccharin sample. The results of Saxholm et al. (1979)
    with the mouse embryo fibroblast test (C3H/10T1/2) and sister
    chromatid exchange, as well as those by Abe & Sasaki (1977) and Wolfe
    & Rodin (1978), are somewhat divergent, possibly due to use of
    saccharins with different purities or the use of different endpoints.
    Simmon et al. (1982) reported on the results of the two independent
    laboratories which attempted to confirm previous short-term positive
    studies when saccharin was introduced into the medium of the following
    tests: mouse lymphoma, human lymphocyte SCE, the CHO cytogenetic
    analyses as well as the standard and modified Salmonella test. As the
    findings from both laboratories were not always similar, the authors
    concluded that the differences observed were attributable in part to
    intrinsic test system variability generated at high concentrations of
    a relatively non-toxic material.

         Various impurities found in Remsen-Fahlberg-produced saccharin
    have been tested in the following systems: ortho- and para-
    toluenesulfonamide were negative with Drosophila (Kramers, 1977) and
    in a modified Salmonella assay (Poncelet et al., 1979, 1980) but
    weakly mutagenic in the Ames Salmonella test and the Basc test in
    Drosophila melanogaster (Eckhardt et al., 1980), while ortho- and
    para-sulfamoylbenzoic acid were ineffective mutagens in the Ames
    Salmonella assay, the Basc test in Drosophila melanogaster and
    micronucleus test in mice (Eckhardt et al., 1980). Such impurities as
    o-sulfobenzoic acid, ammonium o-sulfobenzate, p-sulfobenzoic acid and
    p-sulfamoylbenzoic acid were also negative in a modified Salmonella
    assay (Poncelet et al., 1979, 1980).

    Special studies on pharmacokinetics

         Following the oral administration of 14C-saccharin to pregnant
    rats on the twenty-first day of gestation, Ball et al. (1977) found
    that 0.6% of the administered dose was detectable in the foetuses and
    radioactivity in their urinary bladder was 10 times higher than in
    other tissues 26 hours post-dosing.

         The pharmacokinetic properties of saccharin in man have recently
    been reported. Colburn et al. (1981) had a man (who had not previously
    consumed saccharin on a routine basis) and a woman (who was a regular
    user of saccharin) ingest a single 100 mg dose of saccharin, following
    which blood and urine samples were obtained during the next 24 hours.

    The authors calculated the half-life and apparent volume of
    distribution to be 1.2 and 6.6 hours, and 41.6 and 13.1 litres,
    respectively. While no measurable saccharin was detected in the plasma
    of either subject prior to the test, the female subject was found to
    be excreting 17.4 µg/hour during the 12 hours prior to the start of
    the test. The female subject had chronically used saccharin up to 10
    days prior to the test period. The authors proposed that a deep
    peripheral second compartment existed in the female subject due to her
    chronic use of saccharin. Sweatman et al. (1981) administered
    saccharin to 3 adult male subjects and periodically determined the
    concentration of saccharin during the next 400 minutes after oral
    (2 g) and i.v. (10 mg/kg) doses. The authors found that their i.v.
    data fitted a two-compartment open model with the half-life of the
    second phase being 70 minutes. Following oral ingestion, the decrease
    in plasma saccharin levels was more complex; however, if the same dose
    of saccharin was given after a meal, the peak plasma levels were lower
    and were delayed by 100-120 minutes versus the i.v. dosing situation.
    After 96 hours, 90% of the latter dose had been recovered in the urine
    and 8% in the faeces.

    Special studies on reproduction


         Groups of 21 pregnant mice received 40-168 mg/kg bw per day of
    saccharin through the production of 3 successive litters without
    deleterious effect on growth, litter number and pups per litter when
    compared with controls fed sugar. No histological studies were
    performed (Lehmann, 1929).

         A 6-generation study was carried out by Kroes et al. (1977) in
    which mice were fed diets containing 0, 0.2 or 0.5% sodium saccharin
    during the entire study. The criteria of reproductive performance
    were: fertility index; viability index (percentage survival at 5
    days); lactation index (percentage survival at 21 days); body weight
    at 21 days; and number of infertile males. No consistent changes in
    any of these parameters over the 6 generations were observed. Although
    body weight at 21 days was suppressed in some groups of some
    generations, this response was not consistently observed in later
    generations at the same treatment level. Additionally, there was no
    evidence of any teratogenic effects.


         One aspect of a long-term feeding study with saccharin performed
    at the Wisconsin Alumni Research Foundation (WARF) reported by Tisdel
    et al., 1974 involved the mating of rats from the same treatment
    groups, on a 1:1 basis, after they had received one of the following
    diets for 14 weeks: 0, 0.05, 0.5 or 5% saccharin, produced by the
    Remsen-Fahlberg procedure. The mothers continued to receive the test
    diets during mating, gestation and lactation.

         "Pregnant animals were isolated about 5 days before parturition
    and allowed to deliver and care for their young until the litters were
    weaned 21 days after birth. Survival of progeny was recorded through
    day 28."

         "Data recorded for each litter included: identification of
    parents, number of pups born (total, alive and dead); number of
    survivors 4 and 21 days after birth; and weight of survivors through
    28 days of age. The results showed no effect of saccharin on mating
    efficiency, survival of pups born alive, or weight gain of surviving
    offspring. All groups fed saccharin had smaller average litter sizes
    and reduced average percent live births, when compared to controls,
    but neither response appeared to be related to dose level."

         "The differences were not statistically analyzed, but examination
    of data on individual animals shows that the lower average values are
    attributable to extremely poor performance of one or two animals in
    each group, not to a general reduction. The apparent effect is
    therefore probably within the customary experimental variability."

         "Surviving and dead fetuses were examined for gross
    abnormalities; no evidence of teratogenicity was found" (NAS, 1974).

         A 3-generation reproductive study (Taylor & Friedman, 1974;
    Taylor et al., 1980) was carried out in which rats were continuously
    fed diets containing 0, 0.01, 0.1, 1.0, 5.0 and 7.5% sodium saccharin
    produced by the Remsen-Fahlberg procedure. In the F1a animals, the
    male offspring of dams receiving 5.0% or 7.5% saccharin weighed 12-20%
    less than controls and the females weighed 17-29% less than control
    animals, but both sexes were able to overcome some of these initial
    weight differences during a chronic feeding portion of the study. In
    the F2a litters, the fertility and viability indices were unaffected
    by treatment, but average litter size was slightly decreased for dams
    receiving 5.0% or 7.5% sodium saccharin; the survival index, weaning
    index and body weights of these pups were below controls. In the F2b
    litters, only body weight at weaning was lower than controls for these
    same two groups.

         During a 2-generation lifetime feeding study by Arnold et al.
    (1977, 1980), 50 male and 50 female rats were fed one of the following
    diets (tap water ad libitum except where noted): control; 2.5 mg/kg
    ortho-toluene-sulfonamide (o-TS); 25 mg/kg o-TS; 250 mg/kg o-TS plus
    drinking-water containing 1% NH4Cl; or 5% sodium saccharin produced
    by the Maumee procedure, which contained no detectable o-TS. o-TS is a
    major impurity in Remsen-Fahlberg-produced saccharin, and o-TS is a
    known inhibitor of the enzyme carbonic anhydrase (Miller et al.,
    1956). The animals were mated on a 1:1 basis after 3 months on test.
    The only treatment-related effect on reproduction was a significant
    depression in the lactation index in the 2.5 mg/kg o-TS group and a
    significant decrease in the litter size of the 250 mg/kg o-TS group.

    There were no treatment-related effects upon fertility, gestation or
    viability indices or any effect upon the body weight of pups or the
    number of males in the litter.

    Special studies on teratology

         Teratogenic studies with mice (Tanaka, 1964; Lorke, 1969; Kroes
    et al., 1977), rats (Bough et al., 1967; Fritz & Hess, 1968; Lessel,
    1970; Taylor & Friedman, 1974) and rabbits (Bough et al., 1967;
    Klotzsche, 1969; Lessel, 1970; Tanaka et al., 1973) have been negative
    to date.

         Lederer & Pottier-Arnould (1973) fed pregnant female rats diets
    containing 0.3% saccharin throughout gestation. The pups from
    saccharin-treated dams had a 37.9% incidence of lens anomalies versus
    12.4% incidence for the control animals.

    Acute toxicity

    Animal                Route         (mg/kg bw)      Reference

    Mouse                 Oral            17 500        Taylor et al., 1968
                          i.p.             6 300        Taylor et al., 1968
                                          17 500        Tanaka, 1964

    Hamster               Oral (F)         8 700
    (8-day LD50 value)         (M)         7 400        Althoff et al., 1975

    Rat                   Oral         14 200-17 000    Taylor et al., 1968
                          i.p.             7 100        Taylor et al., 1968

    Rabbit                Oral       5 000-8 000 (LD)   Folin & Herter, 1912

    Dog                   i.v.          2 500 (LD)      Becht, 1920

    Short-term studies


         Kennedy et al. (1976) fed groups of 10 male and female
    weanling rats one of the following diets for 13 weeks: (1) control;
    (2) 20 000 ppm (2%) sodium saccharin; (3) 20 000 ppm (2%)
    o-sulfamoylbenzoic acid (o-SABA); (4) 20 000 ppm (2%) ammonium
    o-carboxybenzene sulfonate (A-o-CBS); (5) 100 ppm (0.01%) sodium
    saccharin plus 450 ppm (0.045%) o-SABA and 450 ppm (0.045%) A-o-CBS;
    (6) 500 ppm (0.05%) sodium saccharin plus 2250 ppm (0.225%) o-SABA and

    2250 ppm (0.225%) A-o-CBS; (7) 2000 ppm (0.2%) sodium saccharin plus
    9000 ppm (0.9%) of o-SABA and 9000 ppm (0.9%) A-o-CBS. (A-o-CBS and
    o-SABA are hydrolytic products of saccharin.) Weight gain and feed
    consumption were determined on a weekly basis and behavioural changes
    were looked for. Haematological studies (RBC, total and differential
    WBC, Hgb and haematocrit), clinical blood chemistry (glucose, BUN,
    serum alkaline phosphatase and SGPT) and urinalysis (albumin, glucose,
    microscopic elements, pH and specific gravity) were conducted prior to
    starting the study, at the midpoint and upon termination. Every animal
    was autopsied and examined histologically. The liver, kidney, spleen
    and gonad weights were determined and organ/body weight ratios
    calculated. No consistent or significant changes in any parameter were

         Groups of 14 male and 14 female rats (75-100 g) received either a
    control diet or one containing 0.5% sodium saccharin for 38 days. The
    inclusion of saccharin in the diet depressed body weight gains and
    feed intake, but efficiency of feed utilization was similar to
    controls. Although diarrhoea was a common observation, no overt
    behavioural changes were apparent. Gross and microscopic inflammatory
    and hydropic changes in the liver and kidneys of the saccharin-treated
    group were reported, but the extent of these changes was not described
    (Taylor et al., 1968).

         Groups of 25 rats (5 males and 20 females) were fed diets
    containing 0, 1.0 or 10% saccharin for 36 weeks. Similar groups of
    rats were fed diets containing 0, 0.1 or 1.0% saccharin for a
    lifetime. One female from each group in the second experiment was
    mated and 4 progeny from each litter were fed a diet containing the
    same amount of saccharin as their parents for a lifetime. Growth was
    retarded at the 10% level, but no adverse effects were seen at the
    lower levels upon histological examination of the major organs (Fantus
    & Hektoen, 1923).

         In a 4-week feeding study, Anderson (1979) fed rats diets
    containing 0, 1, 3, 5 or 7.5% sodium saccharin and observed: (1)
    transient diarrhoea in the 5% and 7.5% group; (2) a dose-dependent
    decrease in urinary ammonia; and (3) a decreased faecal odour. These
    observations led the author to hypothesize that diets with high levels
    of sodium saccharin may lead to changes in the intestinal microflora.
    To test the hypothesis, Anderson & Kirkland (1980) fed rats diets
    containing 0 or 7.5% saccharin for 10 days. The saccharin diet
    produced an increased weight of the caecal tissues and contents; did
    not decrease the total number of anaerobic microbes, but did result in
    the inability to detect a specific anaerobic microbe; increased the
    number of aerobic microbes; and reduced the amount of ammonia produced
    from urea by Proteus vulgaris.


         In addition to the rat study cited above, Kennedy et al. (1976)
    also fed the same 7 diets to groups of 3 male and 3 female beagle dogs
    for 16 weeks. In addition to the parameters indicated for the rats,
    the authors also determined SGOT and protein-bound iodine in this
    study. No significant treatment-related effects were observed.

         One male and 1 female dog received 150 mg/day of saccharin in
    their food for 18 months without any adverse effects on weight,
    fertility or other bodily functions. Their pups developed normally
    (Bonjean, 1922).

         When given doses of 175-350 mg/day for 100 days, dogs developed
    hyperaemia of the lungs, liver, myocardium and kidney, as well as
    cloudy swelling of renal glomeruli and convoluted tubules (de Nito,

         Groups of 4 dogs (sex unspecified) received 6 daily doses of
    sodium saccharin (0.065 g/kg dissolved in water) for 11 months via
    intragastric intubation. The dogs were observed daily for survival,
    general physical condition, behaviour, food and water consumption, and
    character and frequency of excreta. The animals were weighed weekly.
    Laboratory tests included renal and hepatic function tests which were
    performed at 0, 1, 2, 4, 9 and 11 months on test. During the second
    half of the study, 1 dog receiving saccharin became anorexic and
    eventually died, but no abnormal organ pathology was observed. After
    10 months of test, all saccharin-treated dogs developed soft, poorly-
    formed stools without frank diarrhoea. Body weight, haemoglobin, total
    leucocyte count, non-protein nitrogen and phenosulfonephthalein
    retention were within normal limits and total erythrocyte count,
    bilirubin and urinary analyses were also unaffected. Gross and
    histological appearance of viscera were normal in the test group
    (Taylor et al., 1968).


         McChesney et al. (1977) reported no pathological or any other
    significant changes in growth, haematology or clinical chemistry in
    groups of 3 rhesus monkeys of each sex receiving 0 (control) or 500 mg
    of saccharin/kg/day and in groups of 2 male and 2 female monkeys
    receiving 20 or 100 mg of saccharin/kg/day 6 days per week for 79
    months. One animal in each experimental group and 2 control animals
    died before termination of the experiment. None of the deaths were
    attributable to the treatment. Metabolic studies conducted on several
    occasions during the test indicated a rapid, almost exclusively
    urinary excretion of unmetabolized saccharin.


         Doses of 1.5-3.0 g of saccharin/day in man caused a persistent
    sweet metallic taste (Carlson et al., 1923). Single doses of 5-10 g
    have been tolerated and even 100 g orally is said to have caused no
    harm. A few non-fatal cases of acute poisoning and allergic response
    have been reported (NAS-NRC, 1955).

         During high-intake balance studies, 3 male volunteers received
    0.3 g of sodium saccharin/day for a maximum of 4 months and 1-1.5 g
    of sodium saccharin/day for a maximum of 2 months. All of the
    administered saccharin was fully accounted for. Seven volunteers
    received 0.15-0.3 g of saccharin/day for 1.3 months without adverse
    effects except for an increased urine output (Folin & Herter, 1912).
    Doses of 90-180 mg of saccharin/day were well tolerated by children
    aged 10-12 years for 13 months (Jessen, 1890). Diabetic patients have
    received as much as 4.8 g daily for 5 months without adverse effect
    (Neumann, 1926a,b) and 0.4-0.5 g/day for 15-24 years without any
    adverse effects (National Academy of Sciences - National Research
    Council, 1955).

         The principal adverse effects that have been reported from
    saccharin are as follows:

    (1) Mild digestive disturbances were noted by Herter & Folin (1911) in
    volunteers ingesting doses of 1-1.5 g of saccharin/day. Loose stools
    were observed in clinical studies when subjects consumed cyclamates
    plus saccharin in doses of about 7 g/day (Berryman et al., 1968). At
    these dosages, the saccharin intake was about 0.7 g/day. Evidence from
    other studies indicates that cyclamate alone at intakes of 5-7 g/day
    may cause loose stools.

    (2) Allergic responses, principally skin reactions of a phototoxic or
    photosensitivity type occur but appear to be of low incidence and, in
    some cases, may have been due to cyclamate being ingested at the same
    time (Fujita et al., 1965; Stritzler & Samuels, 1956; Kingsley, 1966;
    Boros, 1965; Meisel, 1952; Gordon, 1972; Taub, 1972). Some authors
    have suggested that there may be a cross-sensitivity to sulfonylureas
    and similar drugs known to cause phototoxic skin reactions. Contact
    dermatitis and photosensitivity or phototoxic reactions have not been
    noted in persons occupationally exposed to saccharin (NAS, 1974).


         The present Committee reviewed further findings from
    epidemiological studies that did not reveal any evidence for a
    saccharin-associated increase in bladder tumours. Final assessment of
    the epidemiological aspect of saccharin consumption will be made
    following the review of a large-scale study, now in progress but

    nearing completion. The Committee decided to extend the temporary ADI
    of 0-2.5 mg/kg for saccharin to 1984 and required the submission of
    the results of a long-term feeding study in rats and epidemiological


    Estimate of temporary acceptable daily intake for man

    0-2.5 mg/kg bw.


    Required by 1984

    (1) Submission of the results of a long-term feeding study, currently
    in progress.

    (2) Submission of the results of the epidemiological study, currently
    in progress.


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    See Also:
       Toxicological Abbreviations
       Saccharin (FAO Nutrition Meetings Report Series 44a)
       Saccharin (FAO Nutrition Meetings Report Series 48a)
       SACCHARIN (JECFA Evaluation)
       Saccharin  (IARC Summary & Evaluation, Supplement7, 1987)
       Saccharin  (IARC Summary & Evaluation, Volume 22, 1980)