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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION





    SAFETY EVALUATION OF CERTAIN 
    FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES: 42





    Prepared by the Fifty-first meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)





    World Health Organization, Geneva, 1999
    IPCS - International Programme on Chemical Safety

    STEVIOSIDE

    First draft prepared by
    Dr Josef Schlatter
    Swiss Federal Office of Public Health, Switzerland

         Explanation 
         Biological data
              Biochemical aspects 
                   Absorption, distribution, and excretion 
                   Biotransformation
                   Effects on enzymes and other biochemical parameters 
              Toxicological studies 
                   Acute toxicity
                   Short-term studies of toxicity
                   Long-term studies of toxicity and carcinogenicity
                   Genotoxicity 
                   Reproductive toxicity
                   Developmental toxicity
                   Studies on metabolites: Steviol 
                        Absorption, distribution, and excretion 
                        Effects on enzymes and other biochemical
                             parameters 
                        Acute toxicity 
                        Genotoxicity  
                        Developmental toxicity 
                   Special studies 
                        Cariogenicity 
                        Renal function and vasodilatation 
              Observations in humans
         Comments
         Evaluation 
         References 


    1.  EXPLANATION

         Stevioside is a glycoside of the diterpene derivative steviol
    (ent-13-hydroxykaur-16-en-19-oic acid). Steviol glycosides are natural
    constituents of the plant  Stevia rebaudiana Bertoni, belonging to
    the Compositae family. The leaves of  S. rebaudiana Bertoni contain
    eight different steviol glycosides, the major constituent being
    stevioside (triglucosylated steviol), constituting about 5-10% in dry
    leaves. Other main constituents are rebaudioside A (tetraglucosylated
    steviol), rebaudioside C, and dulcoside A.  S. rebaudiana is native
    to South America and has been used to sweeten beverages and food for
    several centuries. The plant has also been distributed to Southeast
    Asia. Stevioside has a sweetening potency 250-300 times that of
    sucrose and is stable to heat. In a 62-year-old sample from a
    herbarium, the intense sweetness of  S. rebaudiana was conserved,
    indicating the stability of stevioside to drying, preservation, and
    storage (Soejarto et al., 1982; Hanson & De Oliveira, 1993).

         Stevioside and its aglycone steviol may act in plants as a
    feeding deterrent, e.g. against the aphid  Schizaphis graminum. The
    EC50 of stevioside was 650 mg/kg; steviol was more active, with an
    EC50 of 150 mg/kg. Steviol lost its deterrent activity after
    acetylation or glycosylation of the C-13 tertiary hydroxy group or
    methylation of the C-19 carboxylic acid substituent, but the activity
    of steviol was not greatly affected by modification of either the C-16
    exomethylene group or its stereochemistry (Nanayakkara et al., 1987).

         The biochemical pathway for the formation of steviol in
     S. rebaudiana is partly known (Kim et al., 1996), and a simple,
    efficient method for the extraction of steviol glycosides has been
    described (Liu et al., 1997). The chemical structure of stevioside
    (Nanayakkara et al., 1987; Suttajit et al., 1993) is shown in Figure
    1.

    FIGURE 1

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

     Rats

         3H-Stevioside (specific activity, 13 or 46 Ci/mg), administered
    by gavage to groups of three to seven Wistar rats at a dose of 125
    mg/kg bw (10-120 Ci/kg bw), was absorbed slowly, a maximal blood
    concentration of 4.8 g/ml being reached by 8 h. At 4 h, the highest
    concentration was found in the caecum (280 g/g stevioside
    equivalent). At 24 h, the concentrations of radiolabel were low in
    most organs, including blood, corresponding to about 2 g/g or ml
    stevioside equivalent, except in liver (5.7 g/g), adrenal gland

    (12 g/g), small intestine (8.8 g/g), caecum (40 g/g), large
    intestine (12 g/g), and fat  (12 g/g). The elimination half-life was
    24 h. At 48 h, 31% of the radiolabel remained in the body. By five
    days, 68% had been excreted into the faeces, 24% in expired air, and
    2.3% in the urine. In bile-duct cannulated rats, biliary excretion was
    low up to 24 h, increasing rapidly thereafter to reach 41% of the dose
    after three days. The authors concluded that stevioside is absorbed
    from the gut very slowly, that enterohepatic circulation occurs in
    rats, and that faecal excretion is the major route of elimination
    (Nakayama et al., 1986).

         131I-Stevioside (specific activity, 3.7 MBq/mg, equivalent to
    100 Ci/mg; 1.1 MBq, 30 Ci, equivalent to 1 mg/kg bw) was injected
    intravenously to male Wistar rats. The radiolabel in plasma decreased
    rapidly, showing rapid distribution in the body. The highest
    concentrations of radiolabel 10 and 120 min after injection were
    observed in the liver (45 and 5% of the injected dose, respectively)
    and the small intestine (18 and 66%). At 120 min after injection, the
    radiolabel eliminated in the bile represented 52% of the original
    dose; that excreted in the faeces and urine 24 h after injection
    represented 35 and 35%, respectively, of the original dose (Cardoso et
    al., 1996). The Committee considered that this study was of limited
    value since introduction of a large 131I atom into stevioside might
    significantly affect its absorption, distribution, metabolism, and
    excretion in bile or urine.

         The renal excretion of stevioside and its effect on the renal
    excretion of several other substances was studied in groups of 10 male
    Wistar rats, which received intravenous infusions of stevioside at
    doses of 4, 8, 12, or 16 mg/kg bw per hour for 30 min, after a control
    period of 30 min. No significant change in inulin clearance was
    observed, but there was a significant increase in  para-aminohippuric
    acid clearance, fractional sodium excretion (FeNa+), urinary flow as
    percent of glomerular filtration rate, and glucose clearance when
    compared with controls at doses greater than 4 mg/kg bw per hour.
    Stevioside clearance was greater than inulin clearance and smaller
    than  para-amino-hippuric acid clearance at all doses tested. The
    authors concluded that stevioside is secreted by the renal tubular
    epithelium and induces diuresis and natriuresis and a fall in renal
    tubular reabsorption of glucose (Melis, 1992a).

    2.1.2  Biotransformation

         Thin-layer chromatography of the intestinal contents, faeces, and
    bile of groups of three to seven Wistar rats given 3H-stevioside
    (specific activity, 13 or 46 Ci/mg) by gavage at a dose of 125 mg/kg
    bw (10-120 Ci/kg bw) revealed that stevioside is decomposed by rat
    caecal flora to steviol and sugars. Stevioside was detected as the
    major component in the stomach 1 h after administration. After 4 h in
    the small intestine, stevioside, steviolbioside (produced by cleavage
    of the glucose moiety at the C-19 position), and steviol accounted for
    7.6, 8, and 7.5% of the radiolabel; in the caecum, these compounds

    accounted for 39, 17, and 5.1% of the radiolabel, respectively. At 24
    h, stevioside was not detectable in the caecum, but steviol and an
    unidentified metabolite accounted for 16 and 68% of the radiolabel,
    respectively. Steviol was found to be the major metabolite in faeces,
    whereas stevioside and the unidentified metabolite were not
    quantifiable. In bile, most of the radiolabel found up to 24 h was on
    the unidentified metabolite, which was probably a steviol conjugate.
    The authors concluded that orally administered stevioside is not
    readily absorbed from the upper part of the small intestine, but
    metabolites, formed primarily by the bacterial flora in the caecum,
    are absorbed from the lower part of the intestine; they also concluded
    that most of the stevioside is excreted as steviol in the faeces
    (Nakayama et al., 1986). The Committee concluded further that the
    faecal steviol could also have arisen from deconjugation of biliary
    conjugates by the gut flora.

         When 2.5 mg/ml stevioside were incubated under anaerobic
    conditions with whole-cell suspensions of bacteria from rat caecum,
    stevioside was completely degraded to steviol within two days. The
    authors concluded that similar degradation of stevioside occurs in
    humans (Wingard et al., 1980).

         Mass spectral analysis of steviol and some analogues revealed
    characteristic patterns reflecting differential stereochemistry and
    variations in the nature of the substituents present. This information
    was used to identify several metabolites of steviol (by gas
    chromatography-mass spectrometry) which are known to produce a
    mutagenic response in  Salmonella typhimurium strain TM677 with
    metabolic activation. After incubation with a 9000   g fraction
    derived from the livers of Aroclor 1254-pretreated rats, unchanged
    steviol was the predominant compound, and nine metabolites were found.
    The major pathways of mammalian metabolism of steviol proved to be
    allylic oxidation and epoxidation. 15alpha-Hydroxysteviol represented
    67% of the metabolites of steviol  in vitro (Compadre et al., 1988). 

         131I-Stevioside (specific activity, 3.7 MBq/mg, equivalent to
    100 Ci/mg; 1.1 MBq, 30 Ci, equivalent to 1 mg/kg bw) was injected
    intravenously to male Wistar rats. The results of reverse-phase
    high-performance liquid chromato-graphy (RP-HPLC) of the bile showed
    that stevioside was degraded  in vivo and that steviol was the major
    metabolite (47% of radiolabel); 37% of the radiolabel was on
    stevioside, and the remaining 15% was on an unidentified metabolite.
    RP-HPLC analysis of urine 90 min after injection showed the presence
    of stevioside and the same unidentified metabolite found in bile, but
    no steviol. The authors concluded that stevioside is metabolized in
    rat liver to steviol, which is excreted through the bile, and that
    similar degradation occurs in humans (Cardoso et al., 1996). The
    Committee concluded that there is an alternative explanation, namely
    that stevioside is secreted into the bile, is degraded to steviol by
    the gut flora, and is resorbed in the lower gut. The Committee
    considered that this study was of limited value since introduction of
    a large 131I atom into stevioside might significantly affect its
    absorption, distribution, metabolism, and excretion in bile or urine.

         Stevioside was perfused at a concentration of 0.2 or 0.5 mmol/L
    (equivalent to 0.16 and 0.4 mg/ml) into rat livers and was
    recirculated for 2 h. The concentration of stevioside remained
    constant throughout the perfusion. The formation of hydrolysis
    products, especially steviol, was investigated chromatographically,
    with negative results. The authors concluded that the reported
    metabolic transformation of intravenously injected 131I-stevioside is
    either a specific characteristic of this derivative or depends on
    factors that are absent in the isolated perfused rat liver
    (Ishii-Iwamoto & Bracht, 1995). The Committee concluded that the most
    likely explanation for the apparent discrepancy is the fact that
    introduction of the large 131I atom into stevioside altered its
    pharmacokinetic behaviour and that stevioside is secreted into the
    bile  in vivo and is degraded to steviol by the gut flora. 

    2.1.3  Effects on enzymes and other biochemical parameters

         Stevioside given to female RCR/Ha mice did not induce glutathione
     S-transferase activity in liver or intestinal mucosa (Pezzuto et
    al., 1986).

         Stevioside (1 mmol/L, equivalent to 0.8 mg/ml) inhibited
    oxidative phosphorylation and the activity of ATPase (50% inhibition),
    succinate oxidase (8% inhibition), and succinate dehydrogenase (10%
    inhibition). No inhibition of NADH-oxidase or L-glutamate
    dehydrogenase activity was seen. The ADP:O ratio was slightly
    decreased. Substrate respiration was increased at low concentrations
    (up to 0.5 mmol/L, equivalent to 0.4 mg/ml) and inhibited at higher
    concentrations (1 mmol/L, equivalent to > 0.8 mg/ml). The authors
    concluded that stevioside acts as a weak uncoupler of oxidative
    phosphorylation (Kelmer-Bracht et al., 1985).

         Stevioside inhibited oxidative phosphorylation in isolated rat
    liver mitochondria. The concentration required for 50% inhibition of
    ATP synthesis was 1.2 mmol/L, equivalent to 0.97 mg/ml (Vignais et
    al., 1966).

         The effect of stevioside, an inhibitor of long-chain fatty acid
    transport, on ketogenesis and on 14C-carbon dioxide production from
    [1-14C]-palmitate (100-300 mol/L) was investigated in isolated and
    haemoglobin-free perfused rat liver. Stevioside (2.5 mmol/L,
    equivalent to 2 mg/ml) inhibited both parameters but had a smaller
    effect on 14C-carbon dioxide production. At 300 mol/L palmitate and
    150 mol/L albumin, ketogenesis was inhibited by 66%, whereas no
    significant inhibition of 14C-carbon dioxide was seen. The authors
    concluded that these results reflect different degrees of saturation
    of the citric acid cycle and the ketogenic pathway and that changes in
    the redox state of the mitochondrial NAD(+)-NADH complex occur after
    infusion of stevioside (Constantin et al., 1991).

         The Committee noted that the concentrations used in the studies
    conducted  in vitro were very high relative to those achieved in
    blood after oral administration, when the major intestinal metabolite
    that enters the circulation is steviol. These studies may therefore be
    of limited significance.

         When single doses of 200 mol/L stevioside, equivalent to
    650 mg/kg bw, were given orally to 24-h-fasted male Wistar rats,
    either alone or simultaneously with fructose, stevioside increased the
    initial glycogen deposition in the liver. When it was given to the
    rats in the drinking-water at 1 or 2 mmol/L, equivalent to 81 and 160
    mg/kg bw, at the beginning of a fasting period of 24 or 48 h,
    increased hepatic glycogen concentrations were found at 48 h
    (1 mmol/L) and at 24 h (2 mmol/L). The authors concluded that
    stevioside stimulates hepatic glycogen synthesis under gluconeogenic
    conditions (Hbler et al., 1994).

         The effect of stevioside on the transport and metabolism of
    D-glucose and D-fructose was investigated in isolated perfused rat
    liver. The maximal exchange rate of D-glucose was 700 mol/L per
    min/ml, and the Km was 38 mmol/L. Stevioside inhibited D-glucose and
    D-fructose transport across the cell membrane. The half-maximal effect
    at 1 mmol/L D-glucose occurred at 0.8 mmol/L stevioside, equivalent to
    0.65 mg/ml. Stevioside had no effect on D-glucose metabolism, except
    to cause transient changes in D-glucose release, which reflected
    changes in the intracellular concentration. D-Fructose consumption,
    however, was specifically affected (half-maximal effect at 2.8 mmol/L,
    equivalent to 2.3 mg/ml), as were all parameters that depend on
    D-fructose transformation: D-glucose production, L-lactate and
    pyruvate production, and extra oxygen uptake. In livers that released
    D-glucose from endogenous glycogen, strong inhibition of transport
    increased the intracellular:extracellular ratio of D-glucose
    concentration. The control values for this ratio, representing an
    average over the total intracellular water space, were all below unity
    (Ishii et al., 1987).

         Stevioside had no effect on gluconeogenesis or oxygen uptake in
    isolated Wistar rat renal cortical tubules at concentrations up to
    3 mmol/L, equivalent to 2.4 mg/ml. The authors concluded that the lack
    of activity was due to the inability of stevioside to penetrate the
    cell membrane (Yamamoto et al., 1985).

         Intravenous infusion of stevioside at 150 mg/ml to male Wistar
    rats at a dose of 100, 150, or 200 mg/kg bw per hour raised the plasma
    glucose concentrations to 110, 140, and 130% of the control value
    during and after infusion. The glucose turnover rate was not altered,
    but glucose clearance was reduced by infusion of 200 mg/kg bw per hour
    stevioside, from 6.5 to 5 ml/min per kg bw. The plasma insulin
    concentration was unchanged. Pretreatment with angiotensin II and
    arginine vasopressin had no effect, while prazosin, an
    alpha-adrenergic blocker, attenuated the hyperglycaemic effect of
    stevioside and infused insulin inhibited it. Oral administration of
    stevioside at 2000 mg/kg bw had no effect on the plasma glucose

    concentration. The authors concluded that the hyperglycaemic effect of
    stevioside was due to an effect on glucose transport across the cell
    (Suanarunsawat & Chaiyabutr, 1997).

         The effects of stevioside at 1 and 5 mmol/L, equivalent to 0.8
    and 4 mg/ml, on intestinal glucose absorption were examined in hamster
    jejunum by the everted sac technique. Glucose absorption was not
    inhibited (Toskulkao et al., 1995a,b).

         Infusion of stevioside at 15 mol/L, equivalent to 12 g/ml, for
    20 min did not significantly alter the arginine-induced secretion of
    insulin or glucagon in the pancreas of male Wistar rats (Usami et al.,
    1980).

         Stevioside inhibited the action of atractyloside, a known
    inhibitor of the adenine nucleotide carrier of mitochondria and in
    consequence an inhibitor of energy metabolism, in isolated perfused
    rat liver. It decreased the effects of atractyloside on glycolysis,
    glycogenolysis, gluconeogenesis, and oxygen uptake. The concentration
    for half-maximal action of stevioside was 0.5 mmol/L, equivalent to
    0.4 mg/ml. The authors concluded that it acts on the outside of the
    cell, as labelled stevioside did not penetrate the cell membranes
    (Ishii & Bracht, 1986).

         Concomitant treatment of Raji cells (human lymphoblastoid cells
    carrying the Epstein-Barr viral genome) with
    12- O-tetradecanoylphorbol 13-acetate (TPA) and stevioside did not
    inhibit the induction of Epstein-Barr virus early antigen by TPA at
    the highest concentration tested: 50 g/ml (18% inhibition) (Okamoto
    et al., 1983a).

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         Studies of the the toxicity of stevioside given as single doses
    to rodents are summarized in Table 1. No lethality was seen within 14
    days after administration, and no clinical signs of toxicity or
    morphological or histopathological changes were found.

         After intravenous administration of stevioside to
    pentobarbital-anaesthetized dogs at a dose of 32.5 mol/L per kg bw
    (equivalent to 26 mg/kg bw), no significant changes were seen in any
    parameters of whole blood, plasma, or renal function, and there was no
    significant alteration of the renal ultrastructure. The authors
    concluded that stevioside is totally devoid of acute extrarenal
    effects (such as hypoxaemia, which could contribute to nephrotoxicity)
    and direct renal effects during the 6-h period following intravenous
    administration (Krejci & Koechel, 1992).

    Table 1. Acute toxicity of stevioside (purity, 96%) given orally to 
    rodents

                                                                            

    Species     Sex               LD50 (g/kg bw)   Reference
                                                                            

    Mouse       Male and female       > 15         Toskulkao et al. (1997)
    Mouse       Male                  >  2         Medon et al. (1982)
    Rat         Male and female       > 15         Toskulkao et al. (1997)
    Hamster     Male and female       > 15         Toskulkao et al. (1997)
                                                                            


    2.2.2  Short-term studies of toxicity

     Rats

         A 13-week study of toxicity was carried out in Fischer 344 rats
    given doses of 0, 0.31, 0.62, 1.25, 2.5, or 5% in the diet (equivalent
    to 160, 310, 630, 1300, and 2500 mg/kg bw per day) to determine the
    appropriate doses for a two-year study of carcinogenicity. The rats
    were randomly allocated to six groups, each consisting of 10 males and
    10 females. None of the animals died during the administration period,
    and there was no difference in body-weight gain between the control
    and treated groups during administration or in food consumption in the
    later part of the study. The activity of lactic dehydrogenase and the
    incidence of single-cell necrosis in the liver were increased in all
    groups of treated males. The authors considered these effects to be
    nonspecific because of the lack of a clear dose-response relationship,
    the relatively low severity, and their limitation to males. Other
    statistically significant differences in haematological and
    biochemical parameters were also considered to be of minor
    toxicological significance. The authors concluded that a concentration
    of 5% in diet was a suitable maximum tolerable dose of stevioside for
    a two-year study in rats (Aze et al., 1991). 

    2.2.3  Long-term studies of toxicity and carcinogenicity

     Rats

         Groups of 45 male and 45 female inbred Wistar rats were given
    diets containing stevioside (purity, 85%) at 0, 0.2, 0.6, or 1.2%
    (equivalent to 100, 300, and 600 mg/kg bw per day) for two years.
    After 6, 12, and 24 months, blood was obtained from the tail vein of
    five male and five female rats in each dose group for haematological
    and clinical biochemical tests. One week later, these rats were housed
    in metabolism cages for urine collection and were then killed for
    further biochemical, pathological, and histopathological examination.
    All surviving animals were killed at two years. Growth, food use and
    consumption, general appearance, and mortality were similar in treated
    and control groups. The mean life span of rats given stevioside was

    not significantly different from that of the controls. No
    treatment-related changes were observed in haematological, urinary, or
    clinical biochemical values at any stage of the study. The incidence
    and severity of non-neoplastic and neoplastic changes were unrelated
    to the concentration of stevioside in the diet. The NOEL was 1.2%,
    equivalent to 600 mg/kg bw per day. The authors suggested that the
    acceptable daily intake of stevioside for humans was 7.9 mg/kg bw per
    day, on the basis of the stevioside consumption of the rats during the
    first three months (the average for males and females being 790 mg/kg
    bw per day) and a safety factor of 100 (Xili et al., 1992).

         Stevioside (purity, 95.6%) was added to powdered diet at
    concentrations of 0, 2.5, or 5% (equal to 0, 970, and 2000 mg/kg bw
    per day for males and 0, 1100, and 2400 for females) and pelleted
    every three months. The doses were selected on the basis of the
    results of the 13-week study and administered to groups of 50 male and
    50 female Fischer 344/DuCrj rats  ad libitum for 104 weeks.
    Thereafter, all of the groups were maintained on basal diet for four
    weeks. All surviving rats were killed at week 108. The body-weight
    gain of treated animals was slightly depressed, and a relationship was
    seen with the dose of stevioside: 2.3 and 4.4% in males at the low and
    high dose and 2.4 and 9.2% in females at the low and high dose. Food
    consumption did not differ between the groups. The final survival rate
    of males at 5% was significantly decreased, with a rate of 60% versus
    78% in controls. The absolute kidney weights were decreased in male
    and female animals at the high dose; however, there was no significant
    histopathological evidence of neoplastic or non-neoplastic lesions
    attributable to treatment in any organ or tissue, except for a
    decreased incidence of mammary adenomas in females and a reduced
    severity of chronic nephropathy in males. The authors concluded that
    stevioside is not carcinogenic in Fischer 344 rats under these
    experimental conditions (Toyoda et al., 1995, 1997). The Committee
    noted that the report of Toyoda et al. (1995) gives data only for
    individual animals, with no summary tables or figures.

         The effects of stevioside on urinary bladder carcinogenesis
    initiated by  N-nitrosobutyl- N-(4-hydroxybutyl)amine was evaluated
    in male Fischer 344 rats given 0.01% of the nitrosamine in their
    drinking-water for four weeks and then 5% stevioside in their diet,
    equivalent to 5000 mg/kg bw per day, for 32 weeks. All surviving rats
    were sacrificed after 36 weeks and examined histologically. Treatment
    with 5% stevioside did not affect the incidence or extent of papillary
    or nodular hyperplasia in nitrosamine-treated rats. No preneoplastic
    or neoplastic lesions of the urinary bladder were observed in rats
    treated with stevioside only. The authors concluded that stevioside
    does not promote bladder carcinogenesis (Hagiwara et al., 1984; Ito et
    al., 1984).

    2.2.4  Genotoxicity

         Studies of the genotoxicity of stevioside are summarized in
    Table 2.


        Table 2. Results of assays for the genotoxicity of stevioside

                                                                                                                              

    End-point                 Test object                          Concentration       Results       Reference
                                                                                                                              

    Reverse mutation          S. typhimurium TA98, TA100           50 mg/platea        Negativeb     Suttajit et al. (1993)
                                                                   (purity, 99%)

    Reverse mutation          S. typhimurium TA97, TA98, TA100,    5 mg/platec         Negative      Matsui et al. (1996a)d
                              TA102, TA104, TA1535, TA1537         1 mg/platee         Negative
                                                                   (purity, 83%)

    Forward mutation          S. typhimurium TM677                 10 mg/platea        Negative      Matsui et al. (1996a)

    Forward mutation          S. typhimurium TM677                 Not specifieda      Negative      Medon et al. (1982)

    Forward mutation          S. typhimurium TM677                 10 mg/platea        Negative      Pezzuto et al. (1985a)

    umu Gene mutation         S. typhimurium TA1535/pSK1002        5 mg/platea         Negative      Matsui et al. (1996a)

    Gene mutation             B. subtilis H17 rec+, M45 rec-       10 mg/disca         Negative      Matsui et al. (1996a)

    Chromosomal aberration    Chinese hamster lung fibroblasts     8 mg/mlc            Negative      Matsui et al. (1996a)
                                                                   12 mg/mle

    Chromosomal aberration    Human lymphocytes                    10 mg/ml            Negative      Suttajit et al. (1993)

    Chromosomal aberration    Chinese hamster lung fibroblasts     12 mg/mlc           Negative      Ishidate et al. (1984)
                                                                   (purity, 85%)
                                                                                                                              

    a   With and without metabolic activation
    b   A positive response towards TA98 was seen without metabolic activation at 50 mg/ml but not at lower concentrations 
        up to 20 mg/ml 
    c   Without metabolic activation
    d   The same results were cited in an earlier abstract (Matsui et al., 1987).
    e   With metabolic activation
    

    2.2.5  Reproductive toxicity

     Hamsters

         Groups of 10 male and 10 female one-month-old golden hamsters
     (Mesocricetus auratus,) were force-fed with stevioside (purity, 90%)
    at 0, 500, 1000, or 2500 mg/kg bw per day daily. Each female was mated
    and allowed to bear three litters during the experiment. Females in
    late gestation and while lactating (one month) received stevioside in
    the drinking-water. Two weeks after the offspring had been weaned, the
    females were mated again. No abnormalities were found in the growth or
    fertility of animals of either sex. All of the males mated females
    efficiently and successfully; the females showed normal four-day
    oestrus cycles and became pregnant after mating. The duration of
    gestation, number of fetuses, and number of offspring were not
    significantly different from those of controls. Forty hamsters of each
    sex from the first and second litters were divided into four groups
    after weaning and force-fed stevioside at the same doses as their
    parents. These animals also showed normal growth and fertility.
    Histological examination of reproductive tissues from animals of all
    three generations revealed no abnormality that could be linked to
    treatment. The authors concluded that stevioside at doses up to 2500
    mg/kg bw per day affected neither growth nor reproduction in hamsters
    (Yodyingyuad & Bunyawong, 1991).

     Rats

         Groups of 11 male Wistar rats were given stevioside (purity, 96%)
    in the diet at 0, 0.15, 0.75, or 3%, equivalent to 0, 150, 750, and
    3000 mg/kg bw per day, for 60 days before and during mating, and
    groups of 11 female Wistar rats received the same diet for 14 days
    before mating and for seven days during gestation. Rats of each sex at
    the highest dose had slightly decreased body-weight gain. There was no
    treatment-related effect on mating performance or fertility, and no
    malformations were seen in the fetuses. The authors concluded that
    stevioside had no adverse effect on fertility or on the development of
    fetuses (Mori  et al., 1981). The Committee noted a slight but not
    statistically significant increase in the number of dead or resorbed
    fetuses at the highest dose.

         A decoction of 5 g dry  S. rebaudiana in 100 ml water was given
    orally to inbred, adult female rats for 18 days, resulting in an
    intake of approximately 40 ml/kg bw. They were mated with untreated
    rats during the last six days. Fertility was reduced to 21% of that of
    control rats and remained reduced (47%) after a 50-60-day recovery
    period (Mazzei-Planas & Kuc, 1968). 

         The effects of aqueous  S. rebaudiana extracts corresponding to
    0.67 g dried leaves per ml, given at a dose of 2 ml/rat twice a day
    for 60 days, were studied in prepubertal (25-30 days old) rats. The
    end-points were glycaemia; serum concentrations of thyroxine and
    tri-iodothyronine; available binding sites in thyroid hormone-binding
    proteins; binding of 3H-methyltrienolone (a specific ligand of

    androgen receptors) to prostate cytosol; zinc content of the prostate,
    testis, submandibular salivary gland, and pancreas; water content of
    testis and prostate; body-weight gain; and the final weights of the
    testis, prostate, seminal vesicle, submandibular salivary gland, and
    adrenal. None of these parameters was significantly different from
    those in the control group, with the exception of the seminal vesicle
    weight, which fell by about 60%. The authors concluded that if the
     Stevia extract can decrease fertility in rats, the effect is almost
    certainly not exerted on males (Oliveira-Filho et al., 1989).

    2.2.6  Developmental toxicity

     Rats

         Stevioside (purity, 95.6%) dissolved in distilled water was given
    to four groups of 25 or 26 pregnant Wistar rats by gavage once a day
    on days 6-15 of gestation at doses of 0, 250, 500, or 1000 mg/kg bw.
    The rats were sacrificed on day 20 of gestation, and their fetuses
    were examined for malformations. The end-points examined were maternal
    and fetal body weight, number of live fetuses, sex distribution,
    number of resorptions or dead fetuses, and incidence of malformations.
    No treatment-related effect on general or reproductive toxicity was
    observed up to the highest dose. The authors concluded that orally
    administered stevioside is not teratogenic in rats (Takanaka et al.,
    1991; Usami et al., 1995).

    2.2.7  Studies on metabolites: Steviol

    2.2.7.1  Absorption, distribution, and excretion: Steviol

         Intact or bile-duct ligated rats were given [17-14C]-steviol
    (specific activity, 2.9 Ci/mg, 1.7 Ci/rat, corresponding to
    approximately 3 mg/kg bw) either orally or by intracaecal injection.
    After oral administration, 1.5% of the radiolabel was excreted in the
    urine of intact rats and 96% in that of bile-duct-ligated animals; the
    corresponding amounts in faeces were 96 and 3.3%. After intracaecal
    administration of 14C-steviol to bile-duct ligated rats, 94 and 6% of
    the radiolabel was excreted in urine and faeces, respectively. When
    bile was collected over 72 h, all of the intracaecally injected
    radiolabel was recovered. Very little (0.02% of dose) was exhaled as
    14C-carbon dioxide. The authors concluded that steviol is completely
    absorbed from the rat lower bowel (Wingard et al., 1980).

    2.2.7.2  Effects on enzymes and other biochemical parameters: Steviol

         Steviol administered to female RCR/Ha mice did not induce
    glutathione  S-transferase activity in liver or intestinal mucosa
    (Pezzuto et al., 1986).

         Steviol at 0.5 mmol/L, equivalent to 0.16 mg/ml, inhibited
    oxidative phosphorylation and the activity of ATPase (92%), NADH
    oxidase (45%), succinate oxidase (42%), succinate dehydrogenase (46%),
    and L-glutamate dehydrogenase (46%). The ADP:O ratio was decreased.

    Substrate respiration was increased at concentrations up to
    0.5 mmol/L, equivalent to 0.16 mg/ml, and inhibited at > 1 mmol/L,
    equivalent to 0.32 mg/ml. Inhibition of substrate respiration was the
    only effect observed in uncoupled mitochondria. Net proton ejection
    induced by succinate and swelling induced by several substrates were
    inhibited. The authors concluded that steviol acts as a uncoupler of
    oxidative phosphorylation (Kelmer-Bracht et al., 1985).

         Steviol decreased glucose production and inhibited oxygen uptake
    in isolated Wistar rat renal cortical tubules (IC50, 0.3 mmol/L,
    equivalent to 96 g/ml). The authors concluded that this effect is
    consistent with an inhibitory action on oxidative phosphorylation and
    electron transport in mitochondria (Yamamoto et al., 1985).

         Steviol inhibited oxidative phosphorylation in isolated rat liver
    mitochondria. The concentration required for 50% inhibition of ATP
    synthesis was 40 mol/L, equivalent to 13 g/ml. Steviol also
    inhibited the 2,4-dinitro-phenol-stimulated ATPase, phosphorylation of
    exogenous ADP, and exchange between exogenous 14C-ADP and endogenous
    adenine nucleotides. The authors concluded that steviol does not act
    at the level of the coupling mechanism but at the level of
    mitochondrial translocation of adenine nucleotides (Vignais et al.,
    1966).

         The effects of steviol (purity, 90%) on intestinal glucose
    absorption were examined in hamster jejunum by the everted sac
    technique. Thus, 1 mmol/L steviol (equivalent to 318.5 g/ml)
    inhibited glucose absorption by 29-43%, and the inhibition was related
    to the steviol concentration and incubation time. Reductions in the
    intestinal mucosal ATP content and absorptive surface area were
    responsible for the inhibition of glucose absorption. The decrease in
    intestinal mucosal ATP content was accompanied by a decrease in the
    activities of mitochondrial NADH cytochrome c reductase and cytochrome
    oxidase. Steviol did not inhibit the activity of intestinal
    Na+/K+-ATPase or glucose uptake in the intestinal brush-border
    membrane vesicles. Steviol altered the morphology of the intestinal
    absorptive cells. The authors concluded that inhibition of intestinal
    glucose absorption by steviol in hamsters is due to a reduction in
    mucosal ATP content and alteration of the morphology of the intestinal
    absorptive cells (Toskulkao et al., 1995a,b).

         Single doses of 200 mol/L steviol, equivalent to 255 mg/kg bw,
    were given orally to 24-h-fasted male Wistar rats, either alone or
    simultaneously with fructose. Under these conditions, steviol
    increased the initial glycogen deposition in the liver. When steviol
    was given to the rats in drinking-water at 1 or 2 mmol/L, equivalent
    to 32 and 64 mg/kg bw, at the beginning of a fasting period of 24 or
    48 h, it had no effect on hepatic glycogen concentrations (Hbler et
    al., 1994).

         Concomitant treatment of Raji cells (human lymphoblastoid cells
    carrying the Epstein-Barr viral genome) with
    12- O-tetradecanoylphorbol 13-acetate (TPA) and steviol strongly

    inhibited the induction of Epstein-Barr virus early antigen by TPA,
    with 50% inhibition at 25 g/ml (Okamoto et al., 1983a).

         In a study of the effects of steviol at 0.2 mol/L (equivalent to
    64 ng/ml) on the induction of ornithine decarboxylase in mouse skin by
    TPA, the activity in the epidermis had increased by about 300-fold
    4-5 h after application of 17 nmol/L TPA. TPA-induced ornithine
    decarboxylase activity was strongly decreased (63%) when steviol was
    applied to mouse skin 1 h before TPA treatment, concurrently with TPA
    (75%), or 1 h after TPA (71%). Steviol alone did not induce epidermal
    ornithine decarboxylase activity. The authors concluded that steviol
    interferes with the process of induction of this enzyme by TPA in
    mouse skin (Okamoto et al., 1983b).

    2.2.7.3  Acute toxicity: Steviol

         In male and female mice and rats given steviol (purity, 90%)
    orally, the LD50 was > 15 g/kg bw, and 1/15 animals died within 14
    days of administration. The LD50 values in hamsters given steviol
    orally were 5.2 g/kg bw in males and 6.1 g/kg bw in females.
    Histopathological examination of the kidneys revealed severe
    degeneration of the proximal tubular cells, and these structural
    alterations were correlated with increased serum blood urea nitrogen
    and creatinine. The authors concluded that the cause of death was
    acute renal failure (Toskulkao  et al., 1997).

    2.2.7.4  Genotoxicity: Steviol

         Studies of the genotoxicity of steviol are summarized in Table 3.
    The major metabolite of steviol  in vitro, 15alpha-hydroxysteviol,
    was inactive at doses up to 7.5 mg/ml in the forward mutation assay in
     S. typhimurium strain TM677 with metabolic activation.
    15-Oxosteviol, a product of the oxidation of 15alpha-hydroxysteviol,
    was a directly acting mutagen at 25-200 g/ml and was highly toxic to
    bacteria. Moreover, the expression of mutagenicity required the
    presence of the 13-hydroxy group and the C-16 exomethylene group
    (Compadre et al., 1988).

         15-Oxosteviol was not mutagenic in various test systems.
    Repetition of the experiment with  S. typhimurium TM677 failed to
    show significant induction of 8-azaguanine-resistant mutants, even
    when the number of bacteria tested was greatly increased. The authors
    concluded that the earlier positive result reported was due to a
    common mishandling of data obtained in the TM677 system and that
    15-oxosteviol is unlikely to be the active metabolite responsible for
    the mutagenicity of steviol (Procinska et al., 1991).


        Table 3. Results of assays for the genotoxicity of steviol

                                                                                                                                    

    End-point                    Test object                         Concentration           Results       Reference
                                                                                                                                    

    Reverse mutation             S. typhimurium TA98 and TA100       20 mg/platea            Negative      Suttajit et al. (1993)
    Reverse mutation             S. typhimurium TA97, TA98, TA100,    5 mg/platea            Negative      Matsui et al. (1996a)b
                                 TA102, TA104, TA1535, and TA1537    (purity, 99%)

    Forward mutation             S. typhimurium TM677                10 mg/platec            Negative      Matsui et al. (1996a)
                                                                     0.5-10 mg/plated        Positive
    Forward mutation             S. typhimurium TM677                10 mg/platec            Negative      Pezzuto et al. (1985a)
                                                                     10 mg/platee            Positive

    umu Gene mutation            S. typhimurium TA1535/pSK1002       625-1250 g/platec      Positive      Matsui et al. (1996a)
                                                                     1259-2500 g/plated     Positive
    Gene mutation                B. subtilis H17 rec+, M45 rec-      10 mg/disca             Negative      Matsui et al. (1996a)
    Gene mutation                Chinese hamster lung fibroblasts    400 g/mld              Positivef     Matsui et al. (1996a)

    Chromosomal aberration       Chinese hamster lung fibroblasts    0.5 g/mlc               Negative      Matsui et al. (1996a)
                                                                     1-1.5 mg/mld            Positive
    Chromosomal aberration       Human lymphocytes                   0.2 mg/ml               Negative      Suttajit et al. (1993)

    Micronucleus formation       MS/Ae mice                          1000 mg/kg bwg          Negative      Matsui et al. (1996a)
                                                                                                                                    

    a   With and without metabolic activation
    b   The same results are cited in an earlier abstract (Matsui et al., 1987).
    c   Without metabolic activation
    d   With metabolic activation
    e   With metabolic activation derived from phenobarbital- or Aroclor 1254-pretreated rats; fractions from control or 
        3-methylcholanthrene-pretreated rats were ineffective.
    f   Diphtheria toxin-resistant colonies
    g   Toxic: 4/6 mice at highest dose given intraperitoneally died
    

         The expression of the mutagenic activity of steviol in
     S. typhimurium TM677 was dependent on both metabolic activation
    (9000   g fraction derived from phenobarbital- or Aroclor
    1254-pretreated rats) and addition of NADPH. The authors concluded
    that a cytochrome P450 mediates the metabolic activation of steviol to
    a mutagenic species. As partially purified rat liver epoxide hydrolase
    did not inhibit steviol-induced mutagenicity, the authors concluded
    that the active metabolite is not an epoxide.

         A species structurally related to steviol, isosteviol, was not
    active in  S. typhimurium TM677, regardless of whether metabolic
    activation was provided. Similarly, chemical reduction of the
    unsaturated bond linking the carbon-16 and -17 positions of steviol
    resulted in the generation of two isomeric products, dihydrosteviol A
    and B, which were not mutagenic. Ent-kaurenoic acid was also inactive.
    A potential metabolite of steviol, steviol-16alpha,17-epoxide, was
    synthesized chemically and found to be ineffective as a directly
    acting mutagen. The authors concluded that it is a metabolite of an
    integral component of stevioside that is mutagenic. The structural
    features necessary for the expression of mutagenic activity include a
    hydroxy group at position 13 and an unsaturated bond joining the
    carbon atoms at positions 16 and 17 (Pezzuto et al., 1985a, 1986).

         Steviol was mutagenic after metabolic activation in the forward
    mutation assay with  S. typhimurium TM677. The authors confirmed
    first that the 8-aza-guanine resistance of the TM677 mutants resides
    in the chromosomal guanine phosphoribosyltransferase  (gpt) gene,
    since it could be complemented by the  gpt gene of  Escherichia 
     coli. The chromosomal DNA of TM677 and TM677 mutants was digested by
    several restriction enzymes (BamHI, Sau3AI, AluI, TaqI, HaeIII, HpaII,
    and RsaI) and analysed by Southern blot hybridization with a probe for
    the  gpt gene in DNA of  E. coli. No significant difference in DNA
    fragment length was found between the wild type and spontaneous or
    steviol-induced mutants (Matsui et al., 1988, 1989a).

         pSV2-gpt plasmids were treated with metabolically activated
    steviol (concentration not given), and the DNA was subsequently
    analysed by polyacrylamide gel electrophoresis after digestion with
    restriction endonucleases (Sau3AI, HhaI, HpaII). Steviol induced a
    fivefold increase in mutation frequency, and seven mutants were
    obtained, all showing deletions ranging from 20 bp to 2 kb (Matsui et
    al., 1989b).

         Steviol strongly induced mutations at the  gpt gene of  S. 
     typhimurium TM677 when a metabolic activation system was present,
    but it had no activity in reverse mutation assays with  E. coli 
    WP2uvrA/pKM101 or  S. typhimurium TA strains. In order to
    characterize the mutations induced by metabolically activated steviol,
    the chromosomal  gpt alleles of 24 induced (ST clones) and 16
    spontaneous mutants (SP clones) of  S. typhimurium TM677 were
    sequenced, and the mutation spectra were compared. Nine out of 24 of
    the mutations of ST clones were localized in the region between
    nucleotides 280 and 330 from the starting codon ATG, whereas no

    mutations of SP clones were found in that region. The mutations
    identified included transitions (three clones), transversions (four
    clones), a duplication, and a deletion. There were no other marked
    differences between the ST and SP clones: base-change mutations
    predominated over frameshifts and deletions (ST clones, 20 versus
    three; SP clones, 16 versus two), and base-change mutations occurred
    more frequently at G:C pairs than at A:T pairs (ST clones, 15 versus
    five; SP clones, 12 versus four). The authors suggested that
    metabolically activated steviol interrupts DNA synthesis around
    nucleotide 280, thereby stimulating duplication, deletion, and
    untargeted mutagenesis in the defined region of the  gpt gene
    downstream from the site of interruption (Matsui et al., 1996b).

         19- O--D-Glucopyranosyl steviol, a potential hydrolysis product
    of stevioside, was mutagenic to  S. typhimurium TM677 and
    bactericidal in the presence of a metabolic activating system (Pezzuto
    et al., 1986).

         Microsomes derived from human liver mediated a mutagenic response
    of steviol (Pezzuto et al., 1985b).

    2.2.7.5  Developmental toxicity: Steviol

         Groups of 20 pregnant golden hamsters were given steviol (purity,
    90%) at doses of 0, 250, 500, 750, or 1000 mg/kg bw per day (only 12
    animals at the highest dose) by gavage in corn oil on days 6-10 of
    gestation. A significant decrease in body-weight gain and increased
    mortality (1/20, 7/20, and 5/12) were observed at the three highest
    doses, and the number of live fetuses per litter and mean fetal weight
    decreased in parallel. Histopathological examination of the maternal
    kidneys showed a dose-dependent increase in the severity of effects on
    the convoluted tubules (dilatation, hyaline droplets). No
    dose-dependent teratogenic effects were seen. The NOEL was 250 mg/kg
    bw per day for both maternal and developmental toxicity (Wasuntarawat
    et al., 1998).

    2.2.8  Special studies 

    2.2.8.1  Cariogenicity

         Groups of 15 albino Sprague-Dawley rat pups colonized with
     Streptococcus sobrinus received 0.5% stevioside or 30% sucrose in
    the basal diet or basal diet alone and were sacrificed after five
    weeks, when  S. sobrinus was counted and caries were evaluated. There
    was no difference in food or water intake or in weight gain among the
    groups, but significant differences in sulcal caries scores and
     S. sobrinus counts were found between the group receiving sucrose
    and the other groups. There was no significant difference between the
    group receiving stevioside and the controls. The authors concluded
    that stevioside was not cariogenic under the conditions of this study
    (Das et al., 1992).

    2.2.8.2  Renal function and vasodilatation

         The effect of stevioside on renal function was evaluated by
    clearance techniques in groups of seven pentobarbital-anesthetized
    male Wistar rats simultaneously with the effect of indomethacin on the
    renal action of stevioside, given at a priming dose of 4, 8, 12, or
    16 mg/kg bw followed by an infusion rate of 4, 8, 12, or 16 mg/kg bw
    per hour. Mean arterial pressure and renal function were measured.
    Administration of stevioside resulted in a statistically significant,
    dose-related decrease in mean arterial pressure (120  2.3 with 4
    mg/kg bw to 72  4.8 mm Hg with 16 mg/kg bw) and an increase in renal
    plasma flow (10  1.2 with 4 mg/kg bw to 26  2.9 ml/min per kg bw
    with 16 mg/kg bw), with no change in glomerular filtration rate.
    Stevioside also increased fractional sodium (FeNa+) and potassium
    (FeK+) excretion and urine flow (volume/glomerular filtration rate).
    The decrease in mean arterial pressure (control, 120  0.93;
    stevioside, 91  2.5 mm Hg) and increase in renal plasma flow
    (control, 14  1.4; stevioside, 33  2.8 ml/min per kg bw) induced by
    stevioside at 16 mg/kg bw were inhibited by simultaneous
    administration of indomethacin at 2 mg/kg bw, but the glomerular
    filtration rate was not affected. The diuretic, natriuretic, and
    kaliuretic effects of stevioside were also abolished by indomethacin.
    The authors concluded that stevioside behaves like a typical
    vasodilator, causing changes in mean arterial pressure, diuresis,
    natriuresis, and kaliuresis per millilitre of glomerular filtration,
    and that these effects probably depend on prostaglandins (Melis &
    Sainati, 1991a).

         The effects of intravenous administration of verapamil (0.015
    mg/min) and calcium chloride (800 mEq/L, 0.025 ml/kg bw per min) on
    renal function and mean arterial pressure were evaluated in groups of
    10 pentobarbital-anaesthetized male Wistar rats weighing 280-320 g
    during intravenous treatment with stevioside (16 mg/kg bw per min).
    Verapamil significantly increased the hypotensive effect of stevioside
    on mean arterial pressure (control, 120  0.77; stevioside, 96  1.5;
    stevioside plus verapamil, 67  0.70 mm Hg) and on fractional sodium
    excretion (control, 0.76  0.05; stevioside, 1.6  0.10; stevioside
    plus verapamil, 2.7  0.25%). Urinary flow, reported as percent
    glomerular filtration rate, and renal plasma flow were increased
    slightly but not significantly during administration of stevioside
    plus verapamil. In contrast, infusion of calcium chloride into rats
    pretreated with stevioside resulted in a marked attenuation of mean
    arterial pressure (control, 120  1.8; stevioside, 70  1.1;
    stevioside plus calcium chloride, 110  1.6 mm Hg) and renal plasma
    flow (control, 17  3.8; stevioside, 34  2.6; stevioside plus calcium
    chloride, 17  2.9 ml/min per kg bw). The diuresis and natriuresis
    induced by stevioside were also inhibited by simultaneous
    administration of calcium chloride. The authors concluded that
    stevioside acts on arterial pressure and renal function as a calcium
    antagonist, as does verapamil (Melis, 1992b).

         Classical clearance techniques and arterial pressure measurements
    in pentobarbital-anaesthetized male Wistar rats showed that stevioside
    at a priming dose of 8 or 16 mg/kg bw followed by an infusion rate of
    8 or 16 mg/kg bw per h caused a fall in systemic blood pressure and in
    diuresis and natriuresis per millilitre of glomerular filtration rate.
    Verapamil tended to increase the renal and systemic effects of
    stevioside. In contrast, an infusion of calcium chloride into rats
    pretreated with stevioside induced marked attenuation of the
    vasodilatatory responses to stevioside. The authors concluded that
    stevioside, like verapamil, acts as a calcium antagonist (Melis &
    Sainati, 1991b).

         The effect of stevioside (purity, > 90%) at a priming dose of
    16 mg/kg bw followed by an infusion rate of 16 mg/kg bw per h on renal
    function in normal Wistar rats and rats with experimental renal
    hypertension was evaluated by clearance techniques. Stevioside
    provoked hypotension, diuresis, and natriuresis in both groups of
    rats. The normal rats had increased renal plasma flow and a constant
    glomerular filtration rate after stevioside administration, whereas
    the hypertensive rats had increased renal plasma flow and glomerular
    filtration rate. The authors concluded that stevioside impairs a renal
    autoregulation mechanism in this model (Melis, 1992c).

         The effects of administration of aqueous  S. rebaudiana extracts
    corresponding to 0.67 g/ml dried leaves given at 2 ml/rat twice a day
    for 20, 40, or 60 days on renal function and mean arterial pressure
    were studied in normal Wistar rats weighing 80-100 g. Rats treated for
    20 days showed no significant difference from the controls, but
    administration of the crude extract for 40 or 60 days induced
    hypotension, diuresis, and natriuresis, with a constant glomerular
    filtration rate. Increased renal plasma flow was seen only in the
    group treated for 60 days. The authors concluded that oral
    administration of an aqueous extract of dried leaves of  Stevia to
    rats induces systemic and renal vasodilation, causing hypotension,
    diuresis, and natriuresis (Melis, 1995).

         Normotensive and experimentally hypertensive male Wistar rats
    (Goldblatt GII experimental hypertension induced by clipping the left
    renal artery, leaving the contralateral kidney intact) received an
     S. rebaudiana extract corresponding to 0.67 g/ml dried leaves given
    at 2 ml/rat by gavage twice a day (2.7 g dry leaves per day) for 30
    days. Administration of Stevia 10-12 weeks after clipping resulted in
    a significant decrease in mean arterial pressure in both the
    normotensive and hypertensive rats: normotensive, 110  3.0 mm Hg in
    controls versus 70  4.0 mm Hg in those given  Stevia; hypertensive,
    160  3.0 mm Hg in controls versus 110  4.0 mm Hg with  Stevia. The
    glomerular filtration rate was constant in the normotensive rats but
    increased significantly in the hypertensive rats after  Stevia 
    treatment (16  1.3 versus 14  1.3 ml/min per kg bw in the controls
    and  Stevia groups, respectively). Both normotensive and hypertensive
    rats had increased renal plasma flow after administration of  Stevia:
    normotensive, 16  3.1 ml/min per kg bw in controls versus 33  3.2
    ml/min per kg bw in the  Stevia group; hypertensive, 19  2.5 ml/min

    per kg bw in controls versus 37  3.9 ml/min per kg bw in the 
     Stevia group.  Stevia increased urinary flow in both normotensive
    (1.4  0.08% versus 2.3  0.11%) and hypertensive animals (1.5  0.07%
    versus 3.0  0.13%) and also increased sodium excretion
    i(normotensive, 0.61  0.07% in controls versus 1.6  0.2% in the
     Stevia group; hypertensive, 0.70  0.1% in controls versus 2.2 
    0.45% in the  Stevia group). The authors concluded that  Stevia
    impairs renal autoregulation in this model (Melis, 1996).

    2.3  Observations in humans

          S. rebaudiana has been used by Indians in Paraguay as an oral
    contraceptive (Mazzei-Planas & Kuc, 1968; Schvartzman et al., 1977).

         Aqueous extracts of 5 g of  S. rebaudiana leaves were
    administered to 16 volunteers at 6-h intervals for three days, and
    glucose tolerance tests were performed before and after
    administration. Another six volunteers were given an aqueous solution
    of arabinose in order to eliminate possible effects of stress. The
    extract increased glucose tolerance and significantly decreased plasma
    glucose concentrations during the test and after overnight fasting in
    all volunteers (Curi et al., 1986).

    3.  COMMENTS

         After oral administration to rats, stevioside is not readily
    absorbed from the upper small intestine but is hydrolysed to the
    aglycone, steviol, before absorption from the gut. Steviol  per se is
    completely absorbed and is excreted in the bile as conjugates; only a
    very small fraction is detectable in urine. After biliary excretion,
    the conjugates are hydrolysed, and steviol undergoes enterohepatic
    circulation; its elimination half-life is 24 h. Steviol is the only
    faecal metabolite of stevioside that has been identified, and
    excretion in the faeces is the major route. After intravenous
    administration, stevioside is rapidly distributed throughout the body,
    partially secreted by the renal tubular epithelium, and excreted in
    urine.

         At high concentrations, stevioside affected a variety of
    biochemical parameters in rat tissues  in vitro. It weakly inhibited
    oxidative phosphorylation, and steviol was about 30 times more potent
    in this respect. The most likely mechanism is inhibition of the
    mitochondrial translocation of adenine nucleotides. Steviol also
    inhibited glucose absorption from rat gut by reducing the mucosal ATP
    content. Stevioside may also act as a calcium antagonist, showing a
    hypotensive effect and inducing diuresis, natriuresis, and a fall in
    renal tubular reabsorption of glucose. Stevioside may not, however, be
    able to penetrate cell membranes. Although most of these studies were
    performed after intravenous injection of stevioside, orally
    administered extracts of  S. rebaudiana to rats had similar effects
    (hypotension and diuresis). 

         Stevioside has very low acute oral toxicity. Oral administration
    of stevioside at a dietary concentration of 2.5% to rats for two
    years, equal to 970 and 1100 mg/kg bw per day in males and females,
    respectively, had no significant effect. Reduced body-weight gain and
    survival rate were observed at a dietary concentration of 5%
    stevioside. There was no indication of carcinogenic potential in a
    long-term study and no evidence of urinary bladder tumour promoting
    potential in a separate bioassay. 

         In studies of reproductive toxicity, administration of stevioside
    at doses up to 2500 mg/kg bw per day to hamsters and 3000 mg/kg bw per
    day to rats had no effect. Although an aqueous infusion of
     S. rebaudiana administered orally to female rats was reported to
    cause a severe, long-lasting reduction in fertility, the contraceptive
    effect of  Stevia is probably not due to stevioside. Stevioside had
    neither teratogenic nor embryotoxic effects in rats given up to 1000
    mg/kg bw per day by gavage. 

         The results of tests for genotoxicity with stevioside in various
    systems were uniformly negative. 

         The aglycone, steviol, was more acutely toxic than stevioside to
    hamsters but not to rats. Steviol was clearly genotoxic after
    metabolic activation, inducing forward mutations in bacteria and gene
    mutations and chromosomal aberrations in Chinese hamster lung
    fibroblasts. Several mechanistic studies indicated that the structural
    features necessary for the expression of mutagenic activity include a
    hydroxyl group at position 13 and an unsaturated bond joining the
    carbon atoms at positions 16 and 17 of steviol. The fact that
    stevioside is glycosylated at position 13 could explain the absence of
    mutagenicity. The active metabolite of steviol responsible for its
    mutagenic activity is not known. While some data suggest that
    epoxidation may be involved in the metabolic activation of steviol,
    other data indicate that the active metabolite is not an epoxide.
    Preliminary data indicate that human liver microsomes may activate
    steviol to a mutagenic metabolite. 


    4.  EVALUATION

         The Committee noted several shortcomings in the information
    available on stevioside. In some studies, the material tested
    (stevioside or steviol) was poorly specified or of variable quality,
    and no information was available on other constituents or
    contaminants. Furthermore, no studies of human metabolism of
    stevioside and steviol were available. In addition, data on long-term
    toxicity and carcinogenicity were available for stevioside in only one
    species. The mutagenic potential of steviol has been tested
    sufficiently only  in vitro.

         In view of the fact that no information was provided for
    elaboration of specifications for stevioside and that the evaluation
    of the available toxicological data revealed several limitations, the
    Committee was unable to relate the results of the toxicological
    investigations to the article of commerce and could not allocate an
    ADI to stevioside. 

         Before the substance is reviewed again, specifications must be
    developed to ensure that the material tested is representative of the
    material of commerce, and further information should be made available
    on the nature of the substance that was tested, on the human
    metabolism of stevioside, and on the activity of steviol in suitable
    studies of genotoxicity  in vivo .


    5.  REFERENCES

    Aze, Y., Toyoda, K., Imaida, K., Hayashi, S., Imazawa, T., Hayashi, Y.
    & Takahashi, M. (1991) Subchronic oral toxicity study of stevioside in
    F344 rats.  Bull. Natl Inst. Hyg. Sci., 48-54 (in Japanese).

    Cardoso, V.N., Barbosa, M.F., Muramoto, E., Mesquita, C.H. & Almeida,
    M.A. (1996) Pharmacokinetic studies of 131I-stevioside and its
    metabolites.  Nucl. Med. Biol., 23, 97-100.

    Compadre, C.M., Hussain, R.A., Nanayakkara, N.P., Pezzuto, J.M. &
    Kinghorn, A.D. (1988) Mass spectral analysis of some derivatives and
     in vitro metabolites of steviol, the aglycone of the natural
    sweeteners, stevioside, rebaudioside A, and rubusoside.  Biomed. 
     Environ. Mass Spectrom., 15, 211-222.

    Constantin, J., Ishii-Iwamoto, E.L., Ferraresi-Filho, O.,
    Kelmer-Bracht, A.M. & Bracht, A. (1991) Sensitivity of ketogenesis and
    citric acid cycle to stevioside inhibition of palmitate transport
    across the cell membrane.  Braz. J. Med. Biol. Res., 24, 767-771.

    Curi, R., Alvarez, M., Bazotte, R.B., Botion, L.M., Godoy, J.L. &
    Bracht, A. (1986) Effect of  Stevia rebaudiana on glucose tolerance
    in normal adult humans.  Braz.J. Med.  Biol. Res., 19, 771-774
    (English abstract only).

    Das, S., Das, A.K., Murphy, R.A., Punwani, I.C., Nasution, M.P. &
    Kinghorn, A.D. (1992) Evaluation of the cariogenic potential of the
    intense natural sweeteners stevioside and rebaudioside A.  Caries 
     Res., 26, 363-366.

    Hagiwara, A., Fukushima, S., Kitaori, M., Shibata, M. & Ito, N. (1984)
    Effects of three sweeteners on rat urinary bladder carcinogenesis
    initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine.  Gann, 75,
    763-768 (English abstract only).

    Hanson, J.R. & De Oliveira, B.H. (1993) Stevioside and related sweet
    diterpenoid glycosides.  Nat. Prod. Rep., 10, 301-309.

    Hbler, M.O., Bracht, A. & Kelmer-Bracht, A.M. (1994) Influence of
    stevioside on hepatic glycogen levels in fasted rats.  Res. Commun. 
     Chem. Pathol. Pharmacol., 84, 111-118.

    Ishidate, M., Sofuni, T., Yoshikawa, K., Hayashi, M., Nohmi, T.,
    Sawada, M. & Matsuoka, A. (1984) Primary mutagenicity screening of
    food additives currently used in Japan.  Food Chem. Toxicol., 22,
    623-636.

    Ishii, E.L. & Bracht, A. (1986) Stevioside, the sweet glycoside of
     Stevia rebaudiana, inhibits the action of atractyloside in the
    isolated perfused rat liver.  Res. Commun. Chem. Pathol. Pharmacol., 
    53, 79-91.

    Ishii, E.L., Schwab, A.J. & Bracht, A. (1987) Inhibition of
    monosaccharide transport in the intact rat liver by stevioside.
     Biochem. Pharmacol., 36, 1417-1433.

    Ishii-Iwamoto, E.L. & Bracht, A. (1995) Stevioside is not metabolized
    in the isolated perfused rat liver.  Res. Commun. Mol. 
     Pathol.Pharmacol., 87, 167-175.

    Ito, N., Fukushima, S., Shirai, T., Hagiwara, A. & Imaida, K. (1984)
    Drugs, food additives and natural products as promoters in rat urinary
    bladder carcinogenesis. In: Brzsnyi, M., Day, N.E., Lapis, K. &
    Yamasaki, H., eds,  Models, Mechanisms and Etiology of Tumour 
     Promotion (IARC Scientific Publications No. 56), Lyon, International
    Agency for Research on Cancer, pp. 399-407.

    Kelmer-Bracht, A.M., Alvarez, M. & Bracht, A. (1985) Effects of
     Stevia rebaudiana natural products on rat liver mitochondria.
     Biochem. Pharmacol., 34, 873-882.

    Kim, K.K., Sawa, Y. & Shibata, H. (1996) Hydroxylation of
    ent-kaurenoic acid to steviol in  Stevia rebaudiana Bertoni --
    Purification and partial characterization of the enzyme.  Arch. 
     Biochem. Biophys., 332, 223-230.

    Krejci, M.E. & Koechel, D.A. (1992) Acute effects of
    carboxyatractyloside and stevioside, inhibitors of mitochondrial
    ADP/ATP translocation, on renal function and ultrastructure in
    pentobarbital-anesthetized dogs.  Toxicology, 72, 299-313.

    Liu, J., Ong, C.P. & Li, S.F.Y. (1997) Subcritical fluid extraction of
    Stevia sweeteners from  Stevia rebaudiana.  J. Chromatogr.Sci., 35,
    446-450.

    Matsui, M., Matsui, K., Kawasaki, Y., Sawada, M., Nohmi, T., Sofuni,
    T., Yoshihira, K., Ishidate, M., Oda, Y., Noguchi, T., Komatsubara, S.
    & Kitagawa, Y. (1987) Mutagenicity test on stevioside and its
    aglycone, steviol.  Mutat. Res., 182, 366.

    Matsui, M., Matsui, K., Nohmi, T., Mizusawa, H. & Ishidate, M. (1988)
    Mutagenicity of steviol: An analytical approach using a Southern
    blotting system.  Mutat. Res., 203, 377.

    Matsui, M., Matsui, K., Nohmi, T., Mizusawa, H. & Ishidate, M. (1989a)
    Mutagenicity of steviol: An analytical approach using the Southern
    blotting system.  Bull. Natl Inst. Hyg.Sci., 83-87 (in Japanese).

    Matsui, M., Matsui, K., Nohmi, T., Mizusawa, H. & Ishidate, M. (1989b)
    Detection of deletion mutations in pSV2-gpt plasmids induced by
    metabolically activated steviol.  Mutat. Res., 216, 367-368.

    Matsui, M., Matsui, K., Kawasaki, Y., Oda, Y., Noguchi, T., Kitagawa,
    Y., Sawada, M., Hayashi, M., Nohmi, T., Yoshihira, K., Ishidate, M. &
    Sofuni, T. (1996a) Evaluation of the genotoxicity of stevioside and
    steviol using six  in vitro and one  in vivo mutagenicity assays.
     Mutagenesis, 11, 573-579.

    Matsui, M., Sofuni, T. & Nohmi, T. (1996b) Regionally-targeted
    mutagenesis by metabolically-activated steviol: DNA sequence analysis
    of steviol-induced mutants of guanine phosphoribosyltransferase (gpt)
    gene of  Salmonella typhimurium TM677.  Mutagenesis, 11, 565-572.

    Mazzei-Planas, G. & Kuc, J. (1968) Contraceptive properties of
     Stevia rebaudiana. Science, 162, 1007.

    Medon, P.J., Pezzuto, J.M., Hovanec-Brown, J.M., Nanayakkara, N.P.,
    Soejarto, D.D., Kamath, S.K. & Kinghorn, A.D. (1982) Safety assessment
    of some  Stevia rebaudiana sweet principles.  Fed. Proc., 41, 1568.

    Melis, M.S. (1992a) Renal excretion of stevioside in rats.  J. Nat. 
     Prod., 55, 688-690.

    Melis, M.S. (1992b) Influence of calcium on the blood pressure and
    renal effects of stevioside.  Braz. J. Med.Biol. Res., 25, 943-949.

    Melis, M.S. (1992c) Stevioside effect on renal function of normal and
    hypertensive rats.  J. Ethnopharmacol., 36, 213-217.

    Melis, M.S. (1995) Chronic administration of aqueous extract of
     Stevia rebaudiana in rats: Renal effects.  J. Ethnopharmacol., 47,
    129-134.

    Melis, M.S. (1996) A crude extract of  Stevia rebaudiana increases
    the renal plasma flow of normal and hypertensive rats.  Braz. J. 
     Med.Biol. Res., 29, 669-675.

    Melis, M.S. & Sainati, A.R. (1991a) Participation of prostaglandins in
    the effect of stevioside on rat renal function and arterial pressure.
     Braz. J. Med. Biol. Res., 24, 1269-1276.

    Melis, M.S. & Sainati, A.R. (1991b) Effect of calcium and verapamil on
    renal function of rats during treatment with stevioside.  J. 
     Ethnopharmacol., 33, 257-262.

    Mori, N., Sakanoue, M., Takeuchi, M., Shimpo, K. & Tanabe, T. (1981)
    Effect of stevioside on fertility in rats.  J. Food Hyg. Soc. Jpn., 
    22, 409-414 (in Japanese).

    Nakayama, K., Kasahara, D. & Yamamoto, F. (1986) Absorption,
    distribution, metabolism and excretion of stevioside in rats.
     J. Food Hyg. Soc. Jpn., 27, 1-8. 

    Nanayakkara, N.P., Klocke, J.A., Compadre, C.M., Hussain, R.A.,
    Pezzuto, J.M. & Kinghorn, A.D. (1987) Characterization and feeding
    deterrent effects on the aphid,  Schizaphis graminum, of some
    derivatives of the sweet compounds, stevioside and rebaudioside A. 
     J. Nat. Prod., 50, 434-441. 

    Okamoto, H., Yoshida, D. & Mizusaki, S. (1983a) Inhibition of
    12-O-tetradecanoylphorbol-13-acetate-induced induction in Epstein-Barr
    virus early antigen in Raji cells.  Cancer Lett., 19, 47-53. 

    Okamoto, H., Yoshida, D., Saito, Y. & Mizusaki, S. (1983b) Inhibition
    of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine
    decarboxylase activity in mouse epidermis by sweetening agents and
    related compounds.  Cancer Lett., 21, 29-35. 

    Oliveira-Filho, R.M., Uehara, O.A., Minetti, C.A. & Valle, L.B. (1989)
    Chronic administration of aqueous extract of  Stevia rebaudiana 
    (Bert.) Bertoni in rats: Endocrine effects.  Gen. Pharmacol., 20,
    187-191.

    Pezzuto, J.M., Compadre, C.M., Swanson, S.M., Nanayakkara, D. &
    Kinghorn, A.D. (1985a) Metabolically activated steviol, the aglycone
    of stevioside, is mutagenic.  Proc. Natl Acad Sci. USA, 82,
    2478-2482.

    Pezzuto, J.M., Nanayakkara, N.P., Compadre, C.M., Swanson, S.M.,
    Kinghorn, A.D., Guenthner, T.M., Lam, L.K., Sparnins, V.L. &
    Wattenberg, L.W. (1985b) Evaluation of the potential of steviol
    (13-hydroxy-ent-kaurenoic acid) and related diterpenes to mediate
    bacterial mutagenesis and induce glutathione-S-transferase in mice.
     Proc. Am. Assoc. Cancer Res., 26, 94.

    Pezzuto, J.M., Nanayakkara, N.P., Compadre, C.M., Swanson, S.M.,
    Kinghorn, A.D., Guenthner, T.M., Sparnins, V.L. & Lam, L.K. (1986)
    Characterization of bacterial mutagenicity mediated by
    13-hydroxy-ent-kaurenoic acid (steviol) and several
    structurally-related derivatives and evaluation of potential to induce
    glutathione S-transferase in mice.  Mutat. Res., 169, 93-103.

    Procinska, E., Bridges, B.A. & Hanson, J.R. (1991) Interpretation of
    results with the 8-azaguanine resistance system in  Salmonella 
     typhimurium: No evidence for direct acting mutagenesis by
    15-oxosteviol, a possible metabolite of steviol.  Mutagenesis, 6,
    165-167.

    Schvartzman, J.B., Krimer, D.B. & Moreno-Azorero, R. (1977)
    Cytological effects of some medicinal plants used in the control of
    fertility.  Experientia, 33, 663-665.

    Soejarto, D.D., Kinghorn, A.D. & Farnsworth, N.R. (1982) Potential
    sweetening agents of plant origin. III. Organoleptic evaluation of
     Stevia leaf herbarium samples for sweetness.  J. Nat. Prod., 45,
    590-599.

    Suanarunsawat, T. & Chaiyabutr, N. (1997) The effect of stevioside on
    glucose metabolism in the rat.  Can. J. Physiol. Pharmacol., 75,
    976-982.

    Suttajit, M., Vinitketkaumnuen, U., Meevatee U. & Buddhasukh, D.
    (1993) Mutagenicity and human chromosomal effect of stevioside, a
    sweetener from  Stevia rebaudiana Bertoni.  Environ. Health 
     Perspectives, 101, 53-56.

    Takanaka, T., Kawashima, K., Usami, M. & Sakami, K. (1991) A
    teratological study of stevioside administered orally to rats.
    Unpublished report from Department of Pharmacology, Biological Safety
    Research Center, National Institute of Hygienic Sciences, Japan.
    Submitted to WHO by Ministry of Health and Welfare, Food Chemistry
    Division, Japan.

    Toskulkao, C., Sutheerawatananon, M., Wanichanon, C., Saitongdee, P. &
    Suttajit, M. (1995a) Effects of stevioside and steviol on intestinal
    glucose absorption in hamsters.  J. Nutr. Sci. Vitaminol., 41,
    105-113.

    Toskulkao, C., Sutheerawattananon, M. & Piyachaturawat, P. (1995b)
    Inhibitory effect of steviol, a metabolite of stevioside, on glucose
    absorption in everted hamster intestine  in vitro. Toxicol. Lett., 
    80, 153-159.

    Toskulkao, C., Chaturat, L., Temcharoen, P. & Glinsukon, T. (1997)
    Acute toxicity of stevioside, a natural sweetener, and its metabolite,
    steviol, in several animal species.  Drug Chem.Toxicol., 20, 31-44.

    Toyoda, K., Kawanishi, T., Uneyama, C. & Takahashi, M. (1995)
    Re-evaluation of the safety of a food additive (reported in fiscal
    1994). A chronic toxicity/carcinogenicity study of stevioside (a
    substance extracted from  Stevia): Final report. Unpublished report
    from Division of Pathology, Biological Safety Research Center,
    National Institute of Health Sciences, Japan. Submitted to WHO by
    Ministry of Health and Welfare, Food Chemistry Division, Japan.

    Toyoda, K., Matsui, H., Shoda,T., Uneyama, C., Takada, K. & Takahashi,
    M. (1997) Assessment of the carcinogenicity of stevioside in F344
    rats.  Food Chem. Toxicol., 35, 597-603.

    Usami, M., Seino, Y., Takai, J., Nakahara, H., Seino, S., Ikeda, M. &
    Imura, H. (1980) Effect of cyclamate sodium, saccharin sodium and
    stevioside on arginine-induced insulin and glucagon secretion in the
    isolated perfused rat pancreas.  Horm. Metab. Res., 12, 705-706.

    Usami, M., Sakemi, K., Kawashima, K., Tsuda, M. & Ohno, Y. (1995)
    Teratogenicity study of stevioside in rats.  Bull. Natl Inst. Hyg. 
     Sci., 113, 31-35 (in Japanese).

    Vignais, P.V., Duee, E.D., Vignais, P.M. & Huet, J. (1966) Effects of
    atractyligenin and its structural analogues on oxidative
    phosphorylation and on the translocation of adenine nucleotides in
    mitochondria.  Biochim. Biophys. Acta, 118, 465-483. 

    Wasuntarawat, C., Temcharoen, P., Toskulkao, C., Mungkornkarn, P.,
    Suttajit, M. & Glinsukon, T. (1998) Developmental toxicity of steviol,
    a metabilite of stevioside, in the hamster.  Drug Chem.Toxicol., 21,
    207-222.

    Wingard, R.E., Brown, J.P., Enderlin, F.E., Dale, J.A., Hale, R.L. &
    Seitz, C.T. (1980) Intestinal degradation and absorption of the
    glycosidic sweeteners stevioside and rebaudioside A.  Experientia, 
    36, 519-520.

    Xili, L., Chengjiany, B., Eryi, X., Reiming, S., Yuengming, W.,
    Haodong, S. & Zhiyian, H. (1992) Chronic oral toxicity and
    carcinogenicity study of stevioside in rats.  Food Chem. Toxicol., 
    30, 957-965.

    Yamamoto, N.S., Kelmer-Bracht, A.M., Ishii, E.L., Kemmelmeier, F.S.,
    Alvarez, M. & Bracht, A. (1985) Effect of steviol and its structural
    analogues on glucose production and oxygen uptake in rat renal
    tubules.  Experientia, 41, 55-57.

    Yodyingyuad, V. & Bunyawong, S. (1991) Effect of stevioside on growth
    and reproduction.  Hum. Reprod., 6, 158-165.
    


    See Also:
       Toxicological Abbreviations