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    FAO Nutrition Meetings
    Resort Series No. 44A
    WHO/Food Add./68.33




    TOXICOLOGICAL EVALUATION OF SOME
    FLAVOURING SUBSTANCES AND
    NON-NUTRITIVE SWEETENING AGENTS





    Geneva, 21-28 August 1967



    The Eleventh Report of the Joint FAO/WHO Expert Committee on Food
    Additives is published as FAO Nutrition Meetings Report Series,
    1967, No. 44; Wld Hlth Org. techn. Rep. Ser., 1968, 383. This
    Report contains general considerations, including the principles
    adopted for the evaluation, and a summary of the results of the
    evaluations of a number of food additives. Additional information,
    such as biological data and a toxicological evaluation, considered at
    that meeting, is to be found in this document.


    Food and Agriculture Organization of the United Nations
    World Health Organization
    1967


    SACCHARIN

    Chemical name                 o-Benzosulfimide;
                                  2,3-dihydro-3-oxobenzisosulfonazole

    Empirical formula             C7H5NO3S

    Structural formula

    MOLECULAR STRUCTURE 22

    Molecular weight              183.19

    Definition                    Saccharin contains not less than 98 per
                                  cent C7H5NO3S after drying.

    Description                   White crystals or a white, crystalline
                                  powder, odourless, or with a faint,
                                  aromatic odour

    Use                           Non-nutritive sweetener

    CALCIUM SACCHARIN

    Chemical name                 Calcium o-Benzosulfimide; Calcium
                                  salt of 2,3-dihydro-
                                  3-oxobenzisosulfonazole

    Empirical formula             C14H8CaN2O6S2.3-1/2H2O

    Structural formula

    MOLECULAR STRUCTURE 23

    Molecular weight              467.49

    Definition                    Calcium saccharin contains not less
                                  than 95 per cent. C14H8CaN2O6S2
                                  calculated on anhydrous basis.

    Description                   White crystals or a white, crystalline
                                  powder, which is odourless or has a
                                  faint, aromatic, odour and an intensely
                                  sweet taste even in dilute solutions.

    Use                           Non-nutritive sweetener

    SODIUM SACCHARIN

    Chemical name                 Sodium o-Benzosulfamide; Sodium salt
                                  of 2,3-dihydro-3-oxobenzisosulfonazole

    Empirical formula             C7H4NNaO3S.2H2O

    Structural formula

    MOLECULAR STRUCTURE 24

    Molecular weight              241.20

    Definition                    Sodium saccharin contains not less than
                                  98 per cent. and not more than the
                                  equivalent of 101 per cent.
                                  C7H4NNaO3S (205.17) after drying.

    Description                   White crystals or a white, crystalline,
                                  efflorescent powder which is odourless
                                  or has a faint, aromatic odour and an
                                  intensely sweet taste, even in very
                                  dilute solutions.

    Use                           Non-nutritive sweetener

    Biological Data

    Biochemical aspects

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

         Orally administered saccharin appears in the urine of man within
    a half hour and is completely eliminated unchanged in 16-48 hours
    (Staub & Staehelin, 1933), some 90 per cent. being excreted in the
    urine in 24 hours (Folin & Herter, 1912). Intravenous saccharin sodium
    in doses of 2.5 g has been used without adverse effect in sick and
    well people to determine circulation time (Fisher et al., 1933). No
    effect has been noted on human nitrogen balance or protein utilization
    in man by doses up to 4 g/day. Daily doses of 5 g reduced albumin
    absorption and utilization by 0.94 per cent. (Neunann, 1926). No
    abnormal effects on total urinary nitrogen excretion or uric acid
    output was noted by other workers after giving oral saccharin (Folin &
    Herter, 1912). No deleterious effects on blood sugar, kidney function,
    vitamin utilization, blood coagulation or enzyme activity has been
    detected in man (NAS-NRC, 1955). Early observations of hypoglycaemia
    induced by saccharin were confirmed in obese-hyperglycaemic mice by
    feeding 125-175 mg/animal daily for 4 weeks. No effect was shown on
    lean mice and adult rats. Intraperitoneal administration of 100 mg to
    fed rats and 10 mg to fed mice caused hypoglycaemia, but no effect was
    seen in fasted animals even after administration of 1 g (Thompson &
    Mayer, 1959).

         Intravenously administered saccharin sodium appears in the
    thoracic and cervical lymph within a few minutes, is distributed
    throughout all body fluids and appears in saliva and tears.

         One gram of saccharin given in water to goats appeared in the
    milk after 9 hours (Carlson et al., 1923). In vivo perfusion of rat
    stomach and small intestine demonstrated considerable absorption from
    the stomach at pH 1.0 and slow absorption from the small intestine,
    i.e. <9 per cent., in two hours (Kojima et al., 1966).

    Acute toxicity

                                                                       

    Animal         Route      LD50              References
                              (mg/kg 
                              body-weight)
                                                                       
    Mouse          i.p.       17 500            Tanaka, 1964

    Rabbit         oral       5000-8000(LD)     Folin & Herter, 1912

    Dog            i.v.       2500(LD)          Becht, 1920
                                                                       

    Short-term studies

         Dog.    One male and one 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).

         If given doses of 175 mg to 350 mg/day for 100 days, dogs
    developed hyperaemia of lungs, liver, myocardium and kidney as well as
    cloudy swelling of renal glomeruli and convoluted tubules (De Nito,
    1936).

    Teratogenicity and fertility studies

         Mouse. Groups of 21 pregnant mice received 42-168 mg/kg
    body-weight/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). Groups of 4 pregnant mice
    received 125 mg/kg saccharin intragastrically on days 4-5 and 6-7 of
    pregnancy, 250 mg/kg on days 4-5 and 6-7 of pregnancy and 500 mg/kg or
    days 8-10 of pregnancy. Controls received saline. Abnormalities of the
    foetus in terms of resorption or retarded development were seen at 125
    mg/kg and 250 mg/kg but not at 500 mg/kg. The mean foetal LD50 was
    calculated at 155 mg/kg (Tanaka, 1964).

         Rat. Eighteen pregnant controls and 15 pregnant test rats
    received either 0 or 6000 mg/kg/day of saccharin sodium from day 1 to
    20 of pregnancy. Six of the controls and 6 test animals were killed on
    the twenty-first day and no foetal abnormalities were noted. Litter
    size, foetal mortality and foetal weight were unaffected. All test
    animals showed some reduced weight gain during the first week and
    gastric ulceration. The remaining 9 test and 12 control animals were
    allowed to go on to parturition and to suckle their young for 21
    days. Again there was no difference between controls and test animals
    as regards litter size, percentage weaned and weaning weights (Bough
    et al., 1967). In another experiment 2 groups of 10 males and 20
    females were given 0 or 10 000 ppm of saccharin in the diet for 60
    premating days. No effect was seen on libido or fertility. Litter size
    at birth and number of pups weaned were comparable for both groups.
    After 6 weeks without treatment the animals were remated, again
    without difference in litter size and number of pups per litter (Bough
    et al., 1967).

         Rabbit. Oral doses of 0 and 600 mg/kg/day saccharin sodium were
    given to Californian rabbits (8 test and 7 controls) from day 1-29 of
    pregnancy, and the foetuses examined at day 30. The test mothers
    showed reduced body-weight gain in the first week, and gastric
    ulceration. There was no adverse effect on litter size, foetal
    mortality or foetal weight (Bough et al., 1967).

    Carcinogenicity studies

         Saccharin and croton oil together were tested for dermal
    cocarcinogenicity in mice. Although the treated group showed a greater
    incidence of skin papillomata compared with controls, 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 is interpreted as demonstrating a cocarcinogenic effect (Allen et
    al., 1957).

    Long-term studies

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

         In another study, groups of 7-10 male and 9-10 male rats were fed
    0, 0.01, 0.1, 0.5, 1.0 and 5.0 per cent. of saccharin in their diet
    for 2 years. There was slight retardation of growth at the 5 per cent.
    level. All other levels showed no deleterious effect on rate of weight
    gain, mortality, haematology, organ weights or histology (Fitzhugh et
    al., 1951). A further study on groups of 20 male and 20 female rats
    fed 0, 0.005, 0.05, 0.5 and 5.0 per cent. saccharin in their diet for
    2 years included a similar group given 1 ml of an aqueous 1 per cent.
    solution of trypan blue s.c. every 2 weeks for 1 year as a positive
    control. In the 5 per cent. group and the trypan blue group, mortality
    was higher than in the controls. Mortality was lower than for the
    controls in the 0.005 per cent. group. Retardation of growth was
    observed in the males and females of the 5 per cent. group despite
    greater food consumption. There was no difference in the incidence of
    tumours between test and control groups (Lessel, 1967).

    Observations in man

         Doses of 1.5-3.0 g/day in man cause 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/day of saccharin sodium for a maximum of 4 months and 1-1.5
    g/day of saccharin sodium for a maximum of 2 months. All saccharin
    administered was fully accounted for. Seven volunteers received from
    0.15-0.3 g per day of saccharin for 1.3 months without adverse effects
    except for increased urine output (Folin & Herter, 1912). 90-180
    mg/day was well tolerated by children aged 10-12 years for 3 months
    (Jessen, 1890). Diabetic patients have received as much as 4.8 g daily
    for 5 months without adverse effect (Neumann, 1926), and 0.4-0.5 g/day
    for 15-24 years without any adverse effects (NRC, 1955).

    Comments

         The extensive biochemical studies with saccharin and sodium
    saccharin show the inertness of these substances. Following an oral
    dose saccharin appears unchanged in the urine of man within a
    half-hour and is completely excreted within 48 hours. The
    long-recorded use by man without any apparent deleterious effects in
    normal individuals and diabetic patients indicates the safety of the
    normal intakes of saccharin. Although long-term animal studies are
    limited to rats, two reports show no effects at dosage levels as high
    as 1 per cent. and only slight growth retardation at 5 per cent. These
    studies are adequate to rule out carcinogenicity. The cocarcinogenic
    studies are limited to skin application and bladder implantation in
    mice and lack significance in the oral use of saccharin for man.
    Reports on studies in mice, rats and rabbits are adequate to show the
    lack of any effect on fertility and progeny.

    EVALUATION

    Level causing no significant toxicological effects

         Rat: 1 per cent. (10 000 ppm) in the diet, equivalent to 500
    mg/kg body-weight/day.

    Acceptable daily intake for man

                                        mg/kg body-weight

        Unconditional acceptance              0-5

        Conditional acceptance1               5-15

                   

    1 For dietetic foods only

    REFERENCES

    Allen, M. J., Boyland, E., Dukes, C. E., Horning, E. S. & Watson, J.
    G. (1957) Brit. J. Cancer, 11, 212

    Becht, F. C. (1920) J. Pharmacol. exp. Therap., 16, 155

    Bonjean, E. (1922) Rev. Hyg., 44, 50

    Bough, R. G., Lessel, B., Sutton, M. M. & Williams, G. A. H.
    Unpublished report submitted by Boots Pure Drug Co., 1967

    Carlson, A. J., Eldridge, C. J., Martin, H. P. & Foran, F. L. (1923)
    J Metab. Res., 3, 45

    De Nito, G. (1936) Boll. Soc. ital. Biol. sper., 11, 934

    Fantus, B. & Hektoen, L. (1923) J. Amer. Pharm Ass., Sci. Ed., 12,
    318

    Fishberg, A. M., Hitzig,  W. M. & King, F. H. (1933) Proc. Soc. exp 
    Biol. Med., 30, 651

    Fitzhugh, O. G., Nelson, A. A. & Frawley, J. P. (1951) J. Amer.
    Pharm. Ass., Sci. Ed., 60, 583

    Folin, O. & Herter, C. (1912) US Dept. Agric. Rep. No. 94

    Jessen, F. (1890) Arch. f. Hyg., 10, 64

    Kojima, S., Ichibagase, H. & Igudin, S. (1966) Chem. Pharm. Bull.,
    14, 965

    Kusaka, -. (1926) Folia Japon. Pharmakol., 2, 370

    Lehmann, K. B. (1929) Arch. f. Hyg., 101, 39

    Lessel, B. Unpublished report submitted by Boots Pure Drug Co., 1967

    National Academy of Sciences - National Research Council (1955) 
    The safety of artificial sweeteners for use in food, Publication No.
    386

    Neumann, R. O. (1926) Arch. f. Hyg., 263; 97, 275

    Salaman, M. H. & Roe, F. J. C. (1950) Brit. J. Cancer, 10, 363

    Staub, H. & Staehelin, R. (1936) Med. Press Circ., 193, 2

    Tanaka, R. (1964) J. Iwate med. Ass., 16,  330

    Thompson, M. M. & Mayer, J. (1959) Amer. J. Clin. Nutr.,  7, 80
    


    See Also:
       Toxicological Abbreviations
       Saccharin (FAO Nutrition Meetings Report Series 48a)
       Saccharin (WHO Food Additives Series 17)
       SACCHARIN (JECFA Evaluation)
       Saccharin  (IARC Summary & Evaluation, Supplement7, 1987)
       Saccharin  (IARC Summary & Evaluation, Volume 22, 1980)