With the exception of calcium metabisulfite (pyrosulfite) which
    has not previously been evaluated, sulfur dioxide and the other
    sulfites have been evaluated for acceptable daily intake by the Joint
    FAO/WHO Expert Committee on Food Additives in 1961, 1964, 1965 and
    1973 (see Annex I, Refs. 6, 8, 11 and 32). Toxicological monographs
    were issued in 1961, 1964, 1965 and 1973 (see Annex I, Refs. 6, 9, 13
    and 33).

         Since the previous evaluation additional data have become
    available and are summarized and discussed in the following monograph.
    The previously published monographs have been expanded and are
    reproduced in their entirety below.

         At the present time no biological data nor toxicological studies
    conducted with calcium metabisulfite are available.



    Absorption, distribution and excretion

         Sulfite is oxidized in the body to sulfate. Bisulfite reacts
    with aldehydes and ketones, including aldehydic sugars. This is a
    reversible reaction; the equilibrium concentrations depend on
    temperature. The acute effects of sulfite in foods are related to the
    amount and concentration of free sulfur dioxide and to the speed at
    which the additive compounds liberate the bound sulfur dioxide.
    Sulfite may also react reversibly with disulfide linkages in proteins.
    The disulfide is split into one part containing a thiol group and
    another part with an S-sulfonic acid group (Swan, 1957). Four rats
    given oral doses of sodium metabisulfite as a 0.2% Solution eliminated
    55% of the sulfur as sulfate in the urine within the first four hours
    (Bhagat & Locket, 1960). A rapid and quantitative elimination of
    sulfites as sulfate was also observed in man and dog (Rost, 1933).
    Sulfite is a strong inhibitor of some dehydrogenases, e.g. lactate
    dehydrogenase (heart) and malate dehydrogenase; 50% inhibition by
    about 10-5M sulfite (Pfleiderer et al., 1956).

         Small amounts of sulfite are regularly formed in the intermediary
    metabolism of the body in the catabolism of cystine by the non-
    enzymatic decomposition of 8-sulfinyl pyruvic acid to pyruvic acid and
    SO2. The stationary concentration of sulfite in the cells is too
    small to be measured. However, 0.10-0.12 molar equivalent/100 ml was
    found in bull seminal fluid (Larson & Salisbury, 1953). Sulfur dioxide
    is strongly bound by plasma proteins in the form of S-sulfonates.

    These are gradually cleared from the blood but by what mechanism is
    not clear at the present time (Gunnison & Benton, 1971; Gunnison &
    Palmes, 1973). Sulfur dioxide can form complex additive compounds with
    other substances present in foods, for example aldehydes, ketones and
    sugars. The reaction is reversible, the equilibrium being influenced
    by temperature and pH. It also reacts reversibly with disulfide groups
    in proteins. In foods SO2 is therefore present in free and bound
    forms, the bound is the predominating form (Allen & Brook, 1970).
    Following oral administration of 10 or 50 mg SO2/kg (as NaHSO3 mixed
    with Na235SO3), 70-95% of the 35S was absorbed from the intestine
    and voided in the urine of mice, rats and monkeys within 24 hours. The
    majority of the remaining 35S was eliminated in the faeces, the rate
    being species-dependent. Only 2% or less of 35S remained in the
    carcass after one week. Free sulfite was not detected in rat urine
    even after a single oral dose of 400 mg SO2/kg. Neither could
    induction of liver sulfite oxidase be demonstrated either after
    single, or 30 daily doses of 200 mg SO2/kg/day (Gibson & Strong,

    Effects on thiamine

         Treatment of foods with sulfites reduced their thiamine content
    (Morgan et al., 1935; Williams et al., 1935). It has been suggested
    that the ingestion of SO2 in a beverage may effectively reduce the
    level of thiamine in the rest of the diet (Hotzel, 1962). Six rats
    were given a diet providing 40 mg thiamine daily. At weekly intervals,
    an additional 160 mg thiamine was given and the urinary excretion of
    thiamine measured on the following two days. When the response, in
    terms of urinary output of thiamine, appeared to be constant, 160 mg
    thiamine was given together with 120 mg potassium metabisulfite. It
    was found that the addition of SO2 greatly reduced the urinary output
    of thiamine, especially on the day when both were given together
    (Causeret et al., 1965).

         In wine containing 400 ppm (0.04%) SO2, 50% of the thiamine was
    destroyed in one week. However, no loss of thiamine was observed in 48
    hours. The small amounts of SO2 resulting from the recommended levels
    of usage in wine are therefore not likely to inactivate the thiamine
    in the diet during the relatively short period of digestion (Jaulmes,
    1965). In a series of studies Hotzel and co-workers (1969) gave 400 mg
    sulfite/person/day to a group of subjects who were fed on a thiamine
    deficient diet. The diet produced signs of vitamin deficiency in 50
    days. Sulfite dissolved in wine or grape juice was given between days
    15-40. No effect on thiamine status was detected by measurement of
    blood thiamine levels, urinary thiamine excretion, and by
    determination of thiamine-dependent enzyme activity. Clinical,
    neurophysiological, and biochemical investigations produced no
    indication of adverse effects from sulfite. The work of Sharratt
    (1970) also supports the view that SO2 in beverages does not reduce
    the level of thiamine in the rest of the diet.

    Effect on calcium balance

         Interest in this aspect arises from the possibility that sulfate
    formed metabolically from sulfite may serve to increase loss of
    calcium in urine and faeces of man. Levels of 0.5-0.7% calcium
    carbonate in the diet caused increased faecal excretion and diminished
    urinary levels of Ca. Levels up to 0.2% had no effect on the excretion
    of Ca (Causeret & Hugot, 1960). In a further experiment, diets
    containing 0.5 and 1% calcium carbonate and 0.5 and 1% potassium
    metabisulfite (2885 and 5770 ppm (0.2885 and 0.5770%) SO2) were
    administered to young rats and the faecal and urinary excretion of Ca
    measured for 10 days. At the lower level of dietary Ca (0.5%) both
    levels of the metabisulfite caused a significant increase in the
    urinary excretion of Ca but had no effect on the faecal excretion. At
    the higher dietary Ca level (1%) the reverse was found. There was no
    difference between the effects of the two levels of metabisulfite.
    This was interpreted as being due to saturation of the body's capacity
    to convert sulfite to sulfate (Hugot et al., 1965).

         The levels of hepatic vitamin A were determined on both control
    and test rats receiving 1.2 g/litre potassium metabisulfite in the
    drinking-water (700 mg/litre as SO2). There was an insignificant
    decrease in the vitamin A level in the liver of test animals after 10
    days. In another experiment two groups of 40 rats each were kept for
    four months on a diet containing only traces of vitamin A. The
    drinking-water of one group contained 1.2 g/litre potassium
    metabisulfite. Hepatic vitamin A levels were determined at the end of
    each month. A gradual reduction in the liver vitamin A levels was
    observed in both groups. The addition of SO2 to the drinking-water
    did not accentuate this reduction (Causeret et al., 1965).


    Special studies on carcinogenicity Mouse


         Two groups of two-month-old mice (50 males and 50 females/group)
    were given a 1% (1500 mg/kg/day) or 2% (3000 mg/kg/day) potassium
    metabisulfite solution in distilled water ad libitum instead of
    drinking-water for a period of 24 months. Animals of the control group
    (50 males and 50 females) were given distilled water alone. Mice of
    each group received basal diet ad libitum. The animals were
    autopsied at death or at termination of the experiment and major organ
    tissues histologically examined. Mortality was not affected by
    treatment, 94-96% of experimental animals survived beyond 180 days.
    Various kinds of tumours, including leukaemias or lung adenomas, were
    observed in the control group as well as in treated groups. However,

    no significant difference was observed in either incidence of each
    rumour or incidence of all tumours between exposed and control groups
    or between the two exposed groups (Tanaka et al., 1979).

    Special studies on DNA binding

         The possibility that SO2 might cause point mutations was put
    forward by Shapiro et al. (1970) who showed that sulfite can convert
    the nucleic acid base cytosine (which occurs in DNA and RNA) into
    uracil (which is found in RNA only). Hayatsu & Miura (1970) confirmed
    the findings and showed that bisulfite binds to certain nucleotides.
    However, exposure of cells in tissue culture to various concentrations
    of SO2 in the medium showed that strain L cells could tolerate 5 ppm
    (0.0005%) SO2 for periods of eight hours provided a recovery period
    followed each exposure. At higher concentrations, 500-2000 ppm
    (0.05-0.2%) of SO2 there was inhibition of growth; at the 500 ppm
    (0.05%) level the growth was comparable to control cultures. The
    addition of salts of SO2 caused stimulation of growth at lower levels
    and complete inhibition at 2000 ppm (0.2%) NaHSO3 (Thompson & Pace,

    Special studies on mutagenicity

         Using E. coli as an indicator, the frequency of mutation of the
    gene of phage lambda was shown to be increased by a factor of 10 when
    compared with controls, by treatment with 3 M NaHSO3 at Ph 5.6 at
    37°C for one-and-a-half hours (Hayatsu & Miura, 1970). Further studies
    indicated sodium bisulfite specifically induced mutations in only
    those mutants which have cytosine-guanine at the mutant site (Mukai et
    al., 1970). A similar specificity for C:G to A:T (adeninethymine)
    transitions was reported by Summers & Drake (1971) using bacteriophage
    T4rII as a test system (although T4 contains the cytosine analogue
    5-hydroxymethylcytosine). At pH 5.0 inactivation and mutation
    frequency of the phage showed excellent dose-response relationships
    with both sodium bisulfite concentration (0.2-0.9 M) and treatment
    time. The reversion frequency resulting from a four-hour treatment
    with 0.9 M bisulfite was approximately 110 per 107. Recent
    duplication of these experiments, however, revealed the initial
    findings to be in error. Bisulfite was not capable of causing a
    measurable rate of reversion to T4 phage, although a 10- to 20-fold
    lower mutation rate than that initially reported cannot be excluded
    (Drake, 1981).

         No mutagenic activity was exhibited by sodium sulfite (Anon.,
    1975b) or potassium metabisulfite (Anon., 1975c) in in vitro plate
    and suspension microbial assays using Salmonella typhimurium,
    strains TA-1535, TA-1537, and TA-1538, and Saccharomyces cerevisiae,
    either unactivated or activated with liver, lung, or testis
    homogenates from mice, rats, and monkeys. Sodium metabisulfite gives a
    positive response in the microsomal activation test with tester

    strains TA-1535, TA-1537, TA-98 and TA-100 (concentrations of test
    material used not specified) and in the host mediated assay (5 × 108
    cells of tester strains in 1 ml of saline were injected into the
    peritoneal cavity of young male mice, test material administered
    orally, dose levels not defined). Metabisulfite failed to produce such
    an effect in the spot test (Rao & Aiyar, 1975).

         Sodium bisulfite was not mutagenic in the host-mediated assay in
    mice, the dominant lethal assay in rats, or the in vivo cytogenic
    assay in rats at doses up to 150 mg/kg; it showed no mutagenic
    activity on human tissue culture cells in vitro at levels up to
    200 µg/ml (Anon., 1972b). In similar tests conducted on sodium
    metabisulfite by another laboratory (Newell & Maxwell, 1972) no
    mutagenic activity was observed in the host-mediated, dominant lethal,
    or cytogenic assays, but mitotic inhibition and widespread damage to
    anaphase cells were noted when sodium metabisulfite was added to human
    embryonic lung cells growing in tissue culture.

         A study conducted with human embryonic lung cells (WI-38)
    revealed that sodium metabisulfite causes chromosomal aberrations in
    the anaphase stage, but failed to produce positive results in two
    Salmonella typhimurium tester strains (G-46, T 1530) and in the
    yeast strain of Saccharomyces cerevisiae D-3 (conditions of the
    tests were not specified (Green, 1977)).

         Sodium bisulfite induced sister chromatic exchanges (SCEs) in
    Chinese hamster cells in a dose-dependent manner after a two- to
    three-hour exposure at concentrations ranging from 3.0 × 10-5 to
    7.3 × 10-3 M. The frequency of SCE increased approximately threefold
    after a two-hour treatment with a 7 × 10-3 M solution of bi-sulfite.
    Increasing the duration of exposure to this agent from two to 24 hours
    increased the SCE frequency by a factor of approximately 2 (MacRae &
    Stich, 1979). Exposure of Syrian hamster embryo cells (HEC) at natural
    Ph to sodium bisulfite at concentrations of 1-20 mM for 15 minutes
    (incubation temperature 37°C, 11% CO2, six days incubation after
    treatment) caused a dose-dependent increase in the cell transformation
    (DiPaolo et al., 1981). On the other hand, under the same conditions
    of treatment as above the bisulfite was not mutagenic to either
    Chinese hamster V79 or Escherichia coli cells (Mallon & Rossman,

         In a dominant lethality test treatment groups of male rats were
    fed sodium metabisulfite in the diet at dose levels of 0, 125, 416.7
    or 1250 mg/kg/day for a period of 10 weeks, then mated with untreated
    females. No consistent adverse effects were observed with the
    exception of significant reduction of body weight gain in males of
    1250 mg/kg/day group (Food and Drug Administration, 1979).

    Special studies on reproduction


         The effect of sodium bisulfite on differentiating spermatogonia
    has been investigated. Adult mice were given either a single
    intra-peritoneal injection (500, 600, 700, 800, 900 and 1000 mg/kg bw)
    or repeated intraperitoneal injections (200 and 400 mg/kg bw) of
    sodium bisulfite. In the latter case the doses were administered 20,
    30 and 40 times during 28, 42 and 56 days respectively. Different
    types of spermatogonia were enumerated from paraffin sections of
    testis stained with periodic acid - Schiff and Ehrlich's haematoxylin.
    No mortality was observed up to 700 mg/kg dose within 24 hours. At the
    1000 mg/kg dose, 80% of the mice died within 24 hours post-treatment.
    Cytotoxicity data showed that sodium bisulfite, at any of the dosage
    levels tested after acute or repeated administration, did not alter
    the population of various types of spermatogonia (Bhattacharjee et
    al., 1980).


         Six groups of 20 male and 20 female rats were mated (group
    matings) after 21 weeks on diets containing 0, 0.125, 0.25, 0.5, 1.0
    or 2.0% Na2S2O5, 10 males and 10 females being remated at 34 weeks.
    Ten male and 10 female F1a rats were mated at 12 and 30 weeks old to
    give F2a and F2b offspring. Ten males and 15 females of the F2a
    generation were mated at 14 and 22 weeks to give F3a and F3b
    offspring. F1a parents and F2a parents were kept on diets for 104 and
    30 weeks respectively. Incidence of pregnancy, birth weight, and
    postnatal survival were all normal. In the F0 first mating, body
    weight gain of offspring was decreased at 2%, and in F1 matings at 1
    and 2%. The F2 first mating showed decreased weight gain of offspring
    in all test groups at weaning but little effect was seen in offspring
    of the second F2 mating. Litter size was significantly decreased at
    0.5% and above in the first F2 mating only. Body weight of F0 adults
    was unaffected, while F1 females at 2% and F2 males and females at
    2% both showed slight decreased body weight gain (Til et al., 1972b).

    Special studies on teratogenicity

         Teratologic evaluations of intubated sodium bisulfite, sodium
    metabisulfite, and potassium metabisulfite have been made in several
    species. The compounds were administered daily on day 6 through day 15
    of gestation in mice and rats. and day 6 through day 10 in hamsters.
    For sodium bisulfite (Anon., 1972a) the doses in mg/kg bw in mice,
    rats and hamsters were up to 150, 110 and 120 respectively; for sodium
    metabisulfite (Anon., 1972a) in mice, rats and hamsters up to 160, 110
    and 120 respectively; for potassium metabisulfite (Anon., 1975a) in
    mice and rats up to 125 and 155 respectively. In no instance were

    significant effects observed on implantation or on maternal or foetal
    survival. The number of abnormalities found in either the soft or
    skeletal tissues of the test groups did not differ from the number
    occurring spontaneously in the sham-treated controls.

         When sodium metabisulfite was injected in the air cell of
    fertilized eggs at 0 hour, the calculated LD50 (based on an average
    egg weight of 50 g) was 19.5 mg/kg, and when injected after 96 hours
    of incubation the LD50 was 3.4 mg/kg. Injections into the yolk were
    less toxic; at 0 hour the LD50 was 53 mg/kg, and at 96 hours
    162 mg/kg. Significantly elevated levels of abnormalities attributable
    to temporary growth retardation were noted, and a low level of
    structural anomalies involving the head and/or limbs was encountered
    (Anon., 1974). A second laboratory (Hwang & Connors, 1974) tested
    potassium metabisulfite on chick embryos and found it to be quite
    embryotoxic; yolk treatment was more toxic than air cell treatment;
    the evidence was inconclusive with respect to teratological effects. A
    third laboratory (Reid, 1975) found sodium bisulfite to be toxic to
    chick embryos when injected into either the air cell or the yolk with
    greater toxicity following air cell administration. No significant
    teratogenic findings were reported. A fourth laboratory (Verrett,
    1975) found sodium sulfite to be toxic on air cell injection into eggs
    at 0 and 96 hours of incubation (LD50 was 20.7 and 16.7 mg/kg of egg
    respectively) but not significantly toxic on yolk injection. Because
    there were no serious structural abnormalities compared to untreated
    or solvent treated controls it was concluded that sodium sulfite is
    not teratogenic under these conditions.

    Other special studies

         Three-month-old male rats were given water containing 0.9 g
    sodium sulfite (Na2SO3)/litre for one to 10 weeks. During the period
    of 10 weeks animals ingested a total of 41 mmol SO3 per kg body
    weight. Rats were killed at 1, 3, 7 or 10 weeks of treatment (number
    of rats sacrificed each time not stated). Heme synthase and
    glutathione, and RNA were analysed in brain and liver, and brain
    respectively. A significant decrease of heme synthase was observed in
    the brain and liver of rats exposed to sulfite for seven or more weeks
    (Savolainen & Tenhunen, 1982).

    Acute toxicity

                          Solution         LD50
    Animal     Route      conc.      sodium bisulfite   Reference
                          (%)           (mg/kg bw)

    Rat        i.p.       25              498

    Rabbit     i.p.       25              300

    Dog        i.p.       25              244            Wilkins et al.,
    Mouse      i.p.        1.25           675

    Rat        i.p.        5.00           650

    Rat        i.p.        1.25           740

         In rabbits the oral LD50 of sulfite, measured as SO2, was found
    to be between 600 and 700 mg/kg bw (Rost & Franz, 1913).


                            LD50 (mg/kg bw)
    Animal     Route                             Reference
                         Sodium         Sodium
                         bisulfite      sulfite

    Mouse      i.v.        130            175

    Hamster    i.v.         95             -
                                                    Hoppe & Goble, 1951
    Rat        i.v.        115             -

    Rabbit     i.v.         65             -

    Short-term studies


         In thiamine-deficient rats daily oral administration of fruit
    syrup containing 350 ppm (0.035%) of sulfur dioxide in a dose of
    0.5 ml/150 g rat for eight weeks failed to influence growth (Locket,
    1957). Groups of weanling rats numbering five per group were fed 0.6%
    sodium metabisulfite (not less than 3400 ppm (0.340%) as SO2) for six
    weeks. The diets were either freshly sulfited or stored at room
    temperature before use. A reduction in growth occurred in rats
    receiving the fresh diet which was attributed to lack of thiamine.

    Rats fed the diet which had been stored for 75 days developed signs of
    thiamine deficiency and additional toxic effects including diarrhoea
    and stunting of growth which could not be reversed by the
    administration of thiamine (Bhagat & Locket, 1964).

         Three groups of 20-30 rats containing equal numbers of males and
    females received daily doses of sulfite dissolved in water or added to
    wine, and a control group received the same volume of water. The
    levels of sulfite in the two groups receiving wine were equivalent to
    105 mg and 450 mg SO2 per litre respectively and the aqueous solution
    contained potassium metabisulfite equivalent to 450 mg SO2 per litre.
    The effect of this treatment was studied in four successive
    generations, the duration being four months in females and six months
    in males. Groups of animals from the second generation were treated
    for one year. No effect was observed on weight gain, efficiency of
    utilization of protein, biological value of the same protein, or
    reproduction. There was also no effect on the macroscopic or
    microscopic appearance of organs or organ weights. The only effect
    observed was a slight diminution in the rate of tissue respiration by
    liver slices in vitro (Jaulmes, 1964).

         Rats were fed sulfite, as Na2S2O5, in stock or purified diet
    at levels from O.125 to 6% for up to eight weeks. In the preliminary
    study increasing levels of sulfite (0.125-2.0% in the diet) resulted
    in decreased urinary thiamine excretion. Supplementation of the diet
    with 50 mg thiamine/kg prevented the thiamine deficiency as evidenced
    by reduction of offspring mortality, and weight loss to weaning at the
    2% level of sulfite feeding. Toxic manifestations were noted at 1% and
    above (but not at 0.5%) comprising occult blood in the faeces (1% and
    over), reduced growth rate (2% purified diet and 6% purified and stock
    diet), blood in the stomach and anaemia (2% and above), spleen
    enlargement, increased haematopoiesis and diarrhoea (4% and above),
    and increased white blood cells (6%). Histopathological changes in the
    stomach occurred at 1% and over (Til, 1970). Groups of 10 male and 10
    female rats were fed on diets containing 0-8% sodium metabisulfite for
    10-56 days. Vitamin deficiency was prevented by adding thiamine to the
    diet. Diets containing 6% and above depressed food intake and growth
    and caused glandular hyperplasia, haemorrhage, ulceration, necrosis
    and inflammation of the stomach. Anaemia occurred in all animals
    receiving 2% and above and a leucocytosis was observed in those
    receiving 6%. At 4% and above splenic haematopoiesis was found. The
    effects were reversible when sulfite was removed from the diet (Til,

         About 120 rats containing equal numbers of each sex were divided
    into two groups, one receiving potassium metabisulfite equivalent to
    0.6% SO2 in the drinking-water, the other group serving as control.
    No effect was observed after treatment for three months on
    reproduction, mortality or blood count. When the second and third
    generations were treated in the same way for three months the only
    effect observed was a significant reduction in the size of the

    litters of treated mothers. No effect of sulfite on digestive enzymes
    in vitro was observed at a level equivalent to 360 mg SO2 per gram
    of protein. No effect on the incidence of dental caries in the rat was
    produced by 0.5% potassium metabisulfite in the diet. Work is in
    progress on the effects of sulfite on the metabolism of thiamine,
    vitamin A, and calcium (Causeret, 1964).

         Groups of 20 Wistar rats (10 of each sex) were fed diets
    containing 0.125, 0.25, 0.5, 1.0 and 2.0% of sodium hydrogen sulfite
    (770-12 300 ppm (0.077-0.43%) as SO2) for 17 weeks. A group of
    20 rats on untreated diet served as controls. Immediately after
    preparation all diets were stored at -18°C in closed glazed
    earthenware containers for not longer than two weeks. Measurements of
    loss of SO2 on keeping each diet in air for 24 hours at room
    temperature revealed losses amounting to 12.5, 10.0, 14.3, 8.2 and
    2.5% of the sulfite present in the diets as listed above, i.e., with
    increasing SO2 content a decreasing proportion was lost. After 124
    days there was no effect on the growth of male rats. In females, the
    2.0% group grew as well as the controls. Both female groups were used
    for fertility studies, had given birth to litters during the course of
    the test, and had raised their young. The other female groups on lower
    levels of dietary sulfite were not mated and showed significant
    depression of growth (as compared with controls that had been mated).
    Haematological measurements at seven to eight weeks (all groups) and
    at 13 weeks (2% and controls) revealed no effect of sulfite.

         In the diet containing 2% sulfite, thiamine could not be measured
    after 14 days at -18°C; at 1.0 and 0.25% sulfite there was some loss
    of thiamine but this cannot be assessed precisely since the initial
    values are not quoted. Measurements of urinary thiamine excretion
    revealed substantial reduction at one week and particularly at 13
    weeks, in all groups receiving more than 0.125% sulfite in the diet.
    Urine concentration tests were not carried out on a sufficient number
    of animals to permit any firm conclusion to be drawn.

         Males and females of the control and 2% groups were mated with
    rats drawn from the main colony. The only untoward findings with
    females of the 2% group were lower weight of the offspring at seven
    and 21 days of life and 44.3% mortality as compared with mortalities
    of 0, 2.8 and 3.8% in the other groups of young rats. It is claimed
    that no changes were found in relative organ weight (liver, heart,
    spleen, kidneys, adrenalin, testes) nor in microscopical appearance
    (above organs, plus stomach, intestine, uterus, teeth and eyes). Since
    no measure of dispersion is quoted, it is impossible to say whether
    the apparent severe reduction in relative liver weight at the 0.125,
    0.25, 0.5 and 1.O% levels is significant (CIVO Institutes, TNO, 1964).

         Groups of rats (number of rats per group not defined) were fed
    sodium metabisulfite in the diet at levels of 6.0% and 0, 4.0, and
    6.0% for a period of four and 12 weeks respectively (diet was

    supplemented with thiamine, 50 mg/kg of food). In the four-week
    experiment rats were sacrificed at day 4 and at weeks 1, 2, 3 and 4 of
    treatment. In the 12-week experiment rats were killed at eight and 12
    weeks of treatment. After sacrifice the stomach was removed and
    examined for pathological changes. The most striking finding was the
    occurrence of hyperplastic glands in the fundic mucosa in the stomach
    of rats exposed to sulfite for at least a two-week period. Hypoplastic
    glands were exclusively lined by large uniform cells laden with
    acidophilic granules. Light and electron microscopy, as well as enzyme
    histochemistry, showed that the cells lining these glands were
    hyperactive chief cells containing a huge amount of pepsinogen
    granules. A time-sequence study revealed that the hyperactive chief
    cells arise from pre-existing chief cells but are also capable of
    proliferation. The occurrence of glands exclusively lined by chief
    cells is highly unusual since mucous cells rather than chief cells are
    considered to be involved in the regeneration of gastric epithelium
    after physical or chmical damage (Beems et al., 1982).


         One rabbit given 3 g of sodium sulfite by stomach tube each day
    for 185 days lost weight, but all organs were normal post mortem. Two
    rabbits given 1.08 g daily for 127 days gained weight. Autopsy showed
    haemorrhages in the stomach. Three rabbits given 1.8 g daily during
    days 46 and 171 lost weight and autopsy showed stomach haemorrhages
    (Rost & Franz, 1913).


         A dose of 3 g of sodium sulfite daily was given by stomach tube
    to a dog weighing 17 kg for 23 days. Another weighing 34 kg was given
    6-16 g of sodium sulfite daily for 20 days (total dose 235 g). No
    abnormalities were observed on autopsy in the first dog, but the
    second dog had haemorrhages in several organs.

         Sodium sulfite was given by stomach tube to 16 growing dogs in
    daily doses of 0.2-4.8 g for 43-419 days. No damage was observed in
    any of the dogs. Sodium bisulfite was given to two dogs by the same
    method and for the same length of time as in the preceding experiment
    in daily doses of 1.08-2.51 g. Examination of heart, lungs, liver,
    kidney and intestine showed no damage. A total of 91-265 g of sodium
    sulfite fed to five pregnant dogs over a period of 60 days had no
    effect on the weight of the mothers or on the weight gain of the
    litters (Rost & Franz, 1913).


         Groups of 20 castrated male, and 20 female weanling Dutch
    Landrace pigs were placed on diets supplemented with 50 mg/kg
    thiamine, and containing 0, 0.06, 0.16, 0.35, 0.83 or 1.72%
    Na2S2O5. Fourteen males and 14 females/group were sacrificed at

    15-19 weeks and the remainder at 48-51 weeks. In addition, a paired
    feeding study on 15 male and 15 female weanling pigs/group was
    performed for 18 weeks at 0 and 1.72% Na2S2O5. Food intake and
    weight gain were reduced at the 1.72% level, but the pair feeding
    study indicated growth and food conversion were not affected when
    intake was controlled. Mortality was not related to sulfite ingestion.
    Urinary and liver thiamine levels decreased with increasing dose, but
    only at 1.72% were they reduced below the levels found in pigs on
    basal diet alone. Haematology and faecal occult blood determinations
    were comparable in all groups. Organ/body weight ratios were elevated
    at 0.83 and 1.72% for heart, kidney and spleen, and at 1.72% for
    liver. The pair feeding study showed liver and kidney weight ratios to
    be increased at 1.72%. Gross pathology comprised mucosal folds in the
    stomach and black coloration of the caecal mucosa in the top two dose
    levels. At 0.83 and 1.72% histopathological examination showed
    hyperplasia of mucosal glands and surface epithelium in the pyloric
    and cardiac regions. In the pars oesophagea, intraepithelial
    microabscesses, epithelial hyperplasia and accumulations of
    neutrophilic leucocytes in papillae tips were observed. In the caecal
    mucosa macrophages laden with pigment granules (PAS positive
    containing Cu and Fe) were observed at all dose levels, including
    controls. Incidence was markedly increased at 0.83% and above. At
    1.72% fat-containing Kupffer cells were present in usually high
    numbers in the liver (Til et al., 1972a).

         A total of 240 piglets (initial body weight 23.3 kg) divided into
    six groups (20 castrated males and 20 females/group) were fed diets
    containing sodium metabisulfite (Na2S2O5) at levels 0, 0.125, 0.25,
    0.5, 1.0 or 2.0% for a period of 15 and 48 weeks. Fourteen male and 14
    female pigs out of each group were sacrificed after 15 weeks of
    treatment and the remaining animals were sacrificed after 48 weeks of
    treatment. The gastrointestinal tract was removed and grossly and
    microscopically examined. Inflammatory and marked hyperplastic changes
    in the oesophageal region were observed in stomachs of some pigs from
    1 and 2% dose groups of the 48-week exposure period. At the 2% dose
    level in the 15-week exposure period and at 1 and 2% dose levels in
    the 48-week exposure period a number of pigs exhibited hyperplastic
    epithelium in the cardiac and pyloric regions of the stomach. A
    striking black discoloration of the caecal mucosa was noticed in a
    number of pigs of the three highest dose groups in the 48-week
    exposure period. Black discoloration appeared to be due to the
    presence of a considerable number of pigment-laden macrophages within
    the lamina propria. The greenish-black pigment granules contained
    ceroid and copper. The occurrence of ceroid-bearing histiocytes in the
    wall of the digestive tract is a well-known pathological condition in
    man (Feron & Wensvoort, 1973).

    Long-term studies

         Groups of rats numbering from 18 to 24 per group were fed sodium
    bisulfite in dosages of 0.0125, 0.025, 0.05, 0.1, 0.25, 0.5, 1 or 2%
    of the diet for periods ranging from one to two years. The rats fed
    0.05% sodium bisulfite (307 ppm (0.0307%) as SO2) for two years
    showed no toxic symptoms. Sulfite in concentrations of 0.1% (615 ppm
    (0.0615%) as SO2) or more in the diet inhibited the growth of the
    rats, probably through destruction of thiamine in the diet (Fitzhugh
    et al., 1946).

         Three groups of weanling rats containing 18, 13 and 19 animals
    received drinking-water containing sodium metabisulfite at levels of
    0 ppm (0%) SO2, 350 ppm (0.035%) SO2 and 750 ppm (0.075%) SO2.
    Prior interaction of the sulfite with dietary constituents was thus
    prevented. The experiment lasted two and a half years and extended
    over three generations of rats. No effects were observed on food
    consumption, fluid intake, faecal output, reproduction, lactation, or
    the incidence of tumours (Locket & Natoff, 1960).

         A solution containing 1.2 g of potassium metabisulfite per litre
    of water (700 ppm (0.07%) SO2) was administered to 80 weanling rats
    (40 of each sex) over a period of 20 months. A group of 80 rats given
    distilled water served as controls. It was shown that the intake of
    fluid by the test group was the same as that of the controls (but no
    study appears to have been made of SO2 loss from the metabibisulfite
    solution). The intake of SO2 calculated from the consumption of water
    was 30-60 mg/kg bw per day for males and 40-80 mg/kg bw per day for
    females. The following criteria provided no evidence of toxic effect:
    growth rate, food intake, clinical condition, haematological indices
    of blood and bone marrow (except peripheral leucocyte count which was
    increased in males), organ weights (except spleen weight which was
    heavier in females), micropathological examination of a large number
    of tissues, and mortality rate. Fatty change in the liver was mostly
    slight or absent, with a similar incidence and severity in test and
    control groups. Reproduction studies over two generations revealed no
    effect except for a slightly smaller number of young in each litter
    from test animals and a smaller proportion of males in each of these
    litters. Growth of the offspring up to three months was almost
    identical in test and control groups (Cluzan et al., 1965).

         Four groups of 20 rats (10 of each sex on standard diet) were
    given daily doses (30 ml/kg bw) of red wine containing 100 and 450 ppm
    (0.01 and 0.045%) SO2 and aqueous solution of potassium metabisulfite
    (450 ppm (0.045%) SO2) and pure water by oral intubation on six days
    each week for four successive generations. The females were treated
    for four months and the males for six; the second generation was
    treated for one year. The only effect seen was a slight reduction in
    hepatic cellular respiration. All other parameters examined, weight

    gain, weight and macroscopic or histological appearance of various
    organs, appearance and behaviour, proportion of parturient females,
    litter size and weight, biological value of a protein sample, showed
    no changes attributable to SO2 (Lanteaume et al., 1965).

         Groups of 20 male and 20 female rats were fed 0, 0.125, 0.25,
    0.5, 1.0 or 2.0% Na2S2O5 in a diet enriched with 50 ppm (0.005%)
    thimaine for two years. All animals were stressed by breeding at 21
    weeks, and half of each group again at 34 weeks. Percentage loss of
    sulfite from the diet decreased with increasing dietary concentration,
    but increased with increasing time. Thiamine loss increased with
    increasing sulfite concentration. Body weight, food consumption,
    kidney function, and organ weights were all unaffected. Thiamine
    content of urine and liver showed a dose-related decrease commencing
    at 0.125 and 0.25% respectively. However, thimaine levels at 2% were
    comparable to levels in control rats. Marginally reduced haemoglobin
    levels were noted on three occasions in females at 2%, and occult
    blood was noted in faeces at 1% and above. In 10% of the females at
    0.25% and in 10% of the males at 0.5% sulfite, slight indications of
    intestinal blood loss were noted at week 32 only. Pathological changes
    were limited to the stomach (either hyperplasia or inflammation) and
    occurred at 1% and above. Incidence of neoplasms was not increased
    above normal levels at any site at any dose (Til et al., 1972b).

         Groups of rats (number of rats per group not stated) received
    sodium bisulfite (Na2S2O5) in the diet at levels of 0, 0.5, 1.0,
    2.0, 4.0, 6.0 or 8.0% during a period of 10-56 days (short-term test)
    and at levels of 0, 0.125, 0.25, 0.5, 1.0 and 2.0% during a period of
    two years (long-term test). Fed diet was supplemented by thiamine (50
    mg thiamine/kg food). Animals were sacrificed after 10, 28 or 56 days
    of treatment in the short-term test and after 8, 12 or 24 months of
    treatment in the long-term test, and their stomach examined for
    pathological changes. In the forestomach the feeding of sulfite
    induced hyperplastic and inflammatory changes. The hyperplasia mainly
    consisted of hyperkeratosis, acanthosis and papillomatous elevation;
    the inflammatory changes comprised ulcerations and mild cellular
    infiltrates in the submucosa. In the fundic part of the glandular
    stomach the sulfite-induced lesions consisted of haemorrhagic
    microerosions, necrosis of epithelial cells, cellular inflammatory
    infiltrations, and an atypical glandular hyperplasia. These lesions
    were observed predominantly in the stomach of rats fed 4.0, 6.0 or
    8.0% sulfite in the short-term test and 0.5, 1.0, or 2.0% sulfite fed
    in the long-term test. In addition, a mild atrophic gastritis
    developed in about 30% of the rats treated with 2% sulfite for two
    years. A number of neoplastic lesions observed were in the range of
    normal level (Feron & Wensvoort, 1972).


         In man a single oral dose of 4 g of sodium sulfite caused toxic
    symptoms in six of seven persons. In another subject 5.8 g caused
    severe irritation of the stomach and intestine (Rost & Franz, 1913).

         The vomiting reflex in man appeared regularly with doses of
    sulfite equivalent to less than 250 mg SO2, i.e. 3.5 mg SO2
    per kg bw (Lafontaine & Goblet, 1955) (see also under: Effects on

         Sulfur dioxide, while harmless to healthy persons when used in
    recommended concentrations, can induce asthma when inhaled or ingested
    by sensitive subjects, even in high dilution (Freedman, 1980).

         Freedman (1977) reported that out of 272 asthmatic patients 30
    (11%) gave a history of exacerbation of the asthma induced by
    ingestion of orange drinks. Fourteen of these 30 patients were
    challenged by drinking an aqueous solution of SO2 in a concentration
    comparable to that permitted in orange drinks. There was an immediate
    fall in FEV1 (forced-expiratory volume in 1 second) and 10 minutes
    after injection the FEV1 was less than half the pre-ingestion level.
    Of the 14 patients challenged eight reacted with falls in FEV1. A
    rapid onset of bronchospasm was the characteristic response of the
    eight SO2-sensitive patients. Their mean age was 29 years and in
    seven of the eight the asthma was intrinsic.

         A 56-year-old housewife, who suffered from hay fever, bought
    grapes from a street trader before the hay fever season. Immediately
    on eating the grapes she was seized by a severe bout of coughing which
    lasted for several hours. Her non-atopic husband ate the grapes with
    no effect. The grapes were found to contain an excess of SO2. Four
    patients with asthma suffered an exacerbation of symptoms after eating
    grapes purchased in a street market. Analysis again showed an excess
    of SO2 (Freedman, 1980).

         To determine whether subjects with mild asthma or seasonal
    rhinitis have greater bronchomotor responses to sulfur dioxide (SO2)
    than normal subjects, seven asthmatic, seven atopic, and seven normal
    subjects (from 23 to 37 years old) were exposed to either 1, 3 or
    5 ppm (0.0001, 0.0003 or 0.0005%) of SO2 by inhalation during a
    period of 10 minutes and then specific airway resistance (SRaw) was
    measured. The results demonstrated that in the asthmatic subjects SRaw
    increased significantly at all concentrations of SO2, whereas in the
    normal and atopic subjects Sraw increased only at 5 ppm (0.0005%)
    (Sheppard et al., 1980).

         A 23-year-old woman with a history of asthma was challenged
    with a 500 mg capsule (oral ingestion) of metabisulfite. Sodium
    metabisulfite produced severe bronchospasm within 30 minutes of
    ingestion (Baker et al., 1981).


         Sulfites are metabolized and eliminated rather rapidly as the
    sulfate. The effect of sulfite on food components needs further study
    since the earlier observations of toxicity from the feeding of stored
    sulfited foods point to the formation of some toxic addition compound.
    Human studies over short periods showed that 400 mg/day produced no
    effect on thiamine excretion.

         Long-term and three-generation studies on rats using
    metabisulfite in a diet with added thiamine showed a no-effect level
    of 0.215% metabisulfite (equivalent to 72 mg/kg bw per day SO2). The
    results of long- and short-term studies demonstrated that the feeding
    of sulfites at dietary level of 0.5% and above causes pathological
    changes in the stomach.

         While it has been shown that sodium bisulfite reacts with DNA and
    produces mutation in bacteria, the relevance of these observations to
    man is highly questionable. The mutagenicity of bisulfite in bacteria
    is manifest only at low Ph. It has been shown that maximal mutagenic
    activity of bisulfite occurs at pH 5.0 while at pH 7 and 8 no
    induction of mutants was observed. The results of the additional
    mutagenicity studies were inconsistent. Sulfites give a positive and
    negative response in microbial systems, cause chromosomal aberrations,
    sister chromatid exchange (SCE), and cell transformations in the
    mammalian systems. The dominant lethality test was negative. A long-
    term carcinogenicity study in mice with potassium metabisulfite was
    negative. Teratogenicity studies in rodents and chicken embryo failed
    to show any compound-related embryotoxic and teratogenic effects.
    Allergic responses to sulfites have been reported. Although there is
    no biological nor toxicological data available for calcium
    metabisulfite, there is no reason to believe it differs from other
    sulfites (SO2 is the active molecule) when used as a food additive.


    Level causing no toxicological effect

         Rat:   0.125% (1250 ppm) metabisulfite in the diet equivalent to
                70 mg/kg bw per day calculated as SO2.

    Estimate of acceptable daily intake for man

         0-0.7 mg/kg bw*


    *    This ADI is a Group ADI for sulfur dioxide and sulfites expressed
         as sulfur dioxide, covering sodium and potassium metabisulfite,
         sodium sulfite, potassium and sodium hydrogen sulfite and sodium
         thiosulfate. At this time the Group ADI could not be extended to
         calcium metabisulfite because no specifications were available.
         Furthermore, the Committee was not aware of the use of calcium
         metabisulfite as a food additive and requested information in
         this regard.


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    See Also:
       Toxicological Abbreviations
       Sulfur dioxide and sulfites (WHO Food Additives Series 5)
       Sulfur dioxide and sulfites (WHO Food Additives Series 21)