These compounds were evaluated for acceptable daily intake at the
    sixth, eighth, ninth, and seventeenth meetings of the Joint FAO/WHO
    Expert Committee on Food Additives (Annex 1, references 6, 8, 11, and
    32). The ADI allocated to sulfur dioxide at the seventeenth meeting
    encompassed the sulfur dioxide equivalents arising from sodium
    metabisulfite, potassium metabisulfite, sodium sulfite, and sodium
    hydrogen sulfite. Subsequently, calcium hydrogen sulfite, sodium
    thiosulfate, and potassium hydrogen sulfite have been included in the
    group ADI (Annex 1, references 41, 47, and 62). Toxicological
    monographs were published after the sixth, eighth, ninth, seventeenth,
    and twenty-seventh meetings (Annex 1, references 6, 9, 12, 33, and

         Since the last review, 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.


    Biochemical aspects

         Sulfur dioxide and sulfur (IV) oxoanions in solution undergo
    pH-dependent equilibration reactions between sulfur dioxide, sulfurous
    acid, bisulfite ion, and sulfite ion. At normal physiological pH
    values and concentrations of greater than 1 M, the equilibrium is
    between approximately equal proportions of sulfite and bisulfite while
    at the lower pH of the stomach of fasting humans, the equilibrium is
    essentially between bisulfite ion and free sulfur dioxide
    (Green, 1976).

         Sulfur dioxide reacts with a wide range of food components. It
    forms adducts by reversible action with aldehydes and ketones
    (including reducing sugars, acetaldehyde, quinones, and ketoacids),
    with anthocyanins, and with cysteine residues in proteins. In most
    foods and beverages, the adducts with carbonyl compounds, the
    hydroxysulfonates, comprise most of the bound sulfite, and this
    equilibrium reaction has been studied in detail. In the range pH 1 to
    pH 8 the hydroxysulfonates predominate, while at higher pH values
    dissociation occurs (Burroughs & Sparks, 1973a,b,c; Adachi et al.,

         Stable adducts with alpha, ß-unsaturated carbonyl intermediates
    of the Maillard reaction have also been described (McWeeny et al.,
    1974; Wedzicha & McWeeny, 1974a), and irreversible reactions with
    other intermediates of the non-enzymic browning reactions lead to the
    formation of stable 3-deoxy-4-sulpho-osuloses. These stable products
    may account for much of the sulfite originally added to stored
    dehydrated vegetables (Wedzicha & McWeeny, 1974b, 1975) and may be the
    major end-product of sulfite in jams made from sulfited fruit
    (McWeeny et al., 1980).

         Sulfur dioxide reacts irreversibly with thiamine to yield
    pyrimidine sulfonic acid and 4-methylhydroxyethyl thiazole (Dwivedi &
    Arnold, 1973) and, at high concentrations, may destroy cobalamins via
    the formation of photolabile complexes (Gunnison et al., 1981a;
    Gunnison & Jacobsen, 1983).

         Sulfite forms adducts with nicotinamide adenine dinucleotide
    (NAD), flavins, and with cytosine and uracil, their nucleosides, and
    nucleotides (Gunnison, 1981, Shapiro, 1983).

         A comprehensive monograph on the chemistry of sulfur dioxide in
    foods has been published (Wedzicha, 1984).

         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 meq/100 ml was found in bull
    seminal fluid (Larson & Salisbury, 1953).

         Sulfite is oxidized in vivo to sulfate, catalysed by the enzyme
    sulfite oxidase (sulfite;ferricytochrome C oxidoreductase, EC
    located in the mitochondrial intramembranous space. This enzyme has
    been well-characterized as a dimer with subunits containing a
    molybdenum atom, a cytochrome b5 type of haemoprotein, and a pterin
    cofactor. The enzyme is inhibited by tungstate both in vitro and
    in vivo (Cohen & Fridovich, 1971; Johnson et al., 1980).

         Sulfite oxidase is widely distributed in mammalian tissues, with
    most activity being found in liver, heart, and kidney (Gunnison,
    1981). Comparative studies on sulfite oxidase activity nave been
    carried out in the livers of several species, including rats, rabbits,
    dogs, cattle, monkeys, and man (MacLeod et al., 1961; Johnson &
    Rajagopalan, 1976a,b). In general, sulfite oxidase activity in the
    human is slightly less than in the rhesus monkey and rabbit, and
    substantially less than in the other mammals studied; human liver has
    only 5-10% of the specific sulfite oxidase activity of rat liver. It
    has been estimated that sulfite oxidase in the rat is capable of
    oxidizing 750 mmoles sulfite/kg b.w./day, equivalent to 48 g SO2/kg
    b.w./day (Cohen et al., 1975).

         In assays on normal human liver biopsy samples, 3 subjects had
    sulfite oxidase activity of 1.78 mmoles cytochrome c reduced/ min./g
    protein and a fourth had approximately twice this activity
    (Johnson et al., 1980). Sulfite oxidase activity in cultured
    fibroblasts from normal human subjects was found to be 1.07 nmoles
    cytochrome c reduced/min./mg protein (range 0.75-1.76) (Shih et al.,
    1977) and 2.10 nmoles cytochrome c reduced/min./mg protein (range
    0.75-3.03) (Johnson et al., 1980).

         Sulfite oxidase deficiency can be induced in the rat by inclusion
    of sodium tungstate in drinking water, and sulfite oxidase activity
    can be reduced to any required extent by modifying the dose of
    tungstate (Johnson et al., 1974). Using this model, sulfite
    oxidase-deficient rats were shown to have substantially-increased
    levels of urinary and tissue thiosulfate and S-sulfonates (Gunnison
    et al., 1981a,b,c). Administration of 0.5 mmole sulfite/kg b.w. to
    sulfite oxidase-deficient rats produced levels of S-sulfonates in the
    plasma similar to those produced in normal rats by a dose of 10 mmole
    sulfite/kg b.w.; higher levels of plasma sulfite were observed.
    Intubation of 10 to 20 times as much sulfite to normal rats was
    required to produce a systemic sulfite level equivalent to that of

    sulfite oxidase-deficient rats, although the normal animals had 100
    times the sulfite oxidase activity of the deficient animals
    (Gunnison et al., 1981a). It has been suggested that sulfite
    oxidation is limited by the rate of diffusion into the mitochondria
    (Oshino & Chance, 1975).

         Exogenous sulfite arising from inhalation or ingestion of sulfur
    dioxide is oxidized to a significant extent in the lung and intestine
    before entering systemic circulation. About 50% of a dose of
    radiolabelled sulfite was oxidized by empty rat intestines and a
    larger proportion was oxidized in filled intestines. Oxidation may
    occur in the intestinal wall and/or by the gut microflora
    (Pfleiderer et al., 1968).

         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 & Lockett, 1960). A rapid and
    quantitative elimination of sulfites as sulfate was also observed in
    man and dog (Rost, 1933).

         Following oral administraton of 10 or 50 mg SO2/kg (as NaHSO3
    mixed with Na235SO3), 70 to 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 the
    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. Induction of liver sulfite oxidase was not demonstrated
    either after single or 30 daily doses of 200 mg SO2/kg/day (Gibson &
    Strong, 1973).

         Sulfite administered i.v. was cleared rapidly in the rhesus
    monkey with a half-life of 10 minutes for doses of 0.3 to 0.6 mmole/kg
    b.w. The half-life in man was estimated to be about 15 minutes
    (Gunnison & Jacobsen, 1983).

         As a result of rapid metabolism by sulfite oxidase, sulfite does
    not accumulate in the tissues on chronic administration, but is
    eliminated in the urine mainly as sulfate. In several species, less
    than 10% of the dose administered appeared in the urine as sulfite
    (Gunnison & Palmes, 1978).

         A proportion of the sulfite absorbed is converted to thiosulfate
    following (a) reaction with mercaptopyrnvate (Sörbo, 1957), (b)
    metabolism of cysteine-S-sulfonate (Sörbo, 1958), and (c) reaction
    with thiocystine (Szczepkowski & Wood, 1967). Elevated levels of
    thiosulfate in body fluids are observed in sulfite oxidase deficiency

    (vide supra). The mean daily urinary excretion of thiosulfate in
    normal humans was found to be 31.7 ± 12.8 mmole/day (Sörbo & Ohman,
    1978), but this does not represent the extent of formation of
    thiosulfate, since thiosulfate is further metabolized to sulfate
    (Gunnison et al., 1981c; Skarzynski et al., 1959).

         Non-enzymatic reactions of sulfite with tissue components include
    lysis of disulfide bonds, with the formation of S-sulfonates and
    thiols (Cecil, 1963). Under conditions of sulfite loading, appreciable
    amounts of S-sulfonates may be formed, and cysteine-S-sulfonate has
    been found in urine (Gunnison & Palmes, 1974), while glutathione-
    S-sulfonate has been detected in bovine ocular lenses (Waley, 1959).
    Only interchain disulfide bridges of native proteins undergo
    sulfitolysis (Cecil & Wake, 1962), and the protein S-sulfonates formed
    slowly released sulfite ions in the presence of sulfhydryl compounds
    (Swan, 1959). Sulfites are strongly bound in the form of S-sulfonates
    by plasma proteins and are gradually cleared from the blood by
    mechanisms which are not totally clear (Gunnison, 1981; Gunnison &
    Palmes, 1974).

         Sodium sulfite solutions were administered to Sprague-Dawley rats
    in which the portal vein and vena cava were cannulated for blood
    sampling. Examination of plasma showed the presence of S-sulfonates in
    both pre- and post-hepatic blood, whereas free sulfite was detected
    only in portal blood. The author concluded that the sulfite was
    absorbed and rapidly metabolized by oxidation or the formation of
    S-sulfonates (Wever, 1985).

         The formation of protein S-sulfonates is concentration dependent
    in vitro and in vivo. At a dose level of 2 mmole sulfite/kg
    b.w./day, the plasma of rabbits and rhesus monkeys contained
    measurable concentrations of S-sulfonates, while the plasma of rats
    did not. Parenteral administration of 3.2 mmole sulfite/kg b.w./day
    to rats for 5 consecutive days increased plasma S-sulfonate
    concentrations up to 19-30 nmoles/ml. Plasma S-sulfonate fractions had
    half-lives of 4 and 8 days in the rat and rhesus monkey, respectively
    (Gunnison & Palmes, 1978). Levels of plasma S-sulfonates in human
    subjects exposed to atmospheric sulfur dioxide concentrations of
    0.3, 1.0, 3.0, 4.2, and 6.0 ppm for 120 hours increased by
    1.1 ± 0.16 nmoles/ml for each increment of 1 ppm in exposure level
    (Gunnison & Palmes, 1974).

         Lungs and aortas of rabbits exposed to arterial sulfite
    concentrations of 545-560 µM for 0.6 to 6 hours were analysed for
    S-sulfonates. Formation was first order, with asymptotic
    concentrations of S-sulfonates of approximately 900 and 9000 nmole/g
    dry tissue in lung and aorta, respectively. Clearance from both
    tissues was first order, with a half-life of 2-3 days. Appreciable
    amounts of S-sulfonates were not found in liver, kidneys, testes,
    heart, brain, skeletal muscle, stomach, ovaries, duodenum, spleen, or
    eyes (Gunnison & Farrugella, 1979).

         Levels of S-sulfonates in the tracheas of rabbits exposed to
    3 ppm sulfur dioxide in air for 3 and 24 hours were constant at
    approximately 53 nmoles/g dry weight. Plasma concentrations of
    S-sulfonates in rabbits exposed to 10 ppm sulfur dioxide were found to
    be 9 and 30 nmoles/ml after 3 and 24 hours, respectively. No exogenous
    S-sulfonates were detected in aortas and only traces were detected in
    the distal region of posterior lung lobes, indicating that sulfur
    dioxide was partially metabolized in pulmonary tissues. Tracheal
    concentrations of S-sulfonates of 107 nmoles/g dry weight were
    attained after 3 hours exposure to 10 ppm sulfur dioxide, increasing
    to 163 nmoles/g dry weight after 72 hours, the increase being
    attributed to increased mucus production. During exposure for 72 hours
    plasma concentrations of S-sulfonates rose to 70 nmoles/ml, but no
    free sulfite was detected in plasma. It was concluded that, with the
    possible exception of heart and lungs, there was no transport of
    inhaled sulfur dioxide to organs distant from the absorption site
    (Gunnison et al., 1981c).

         Sulfur dioxide can promote cross-linking of protein molecules,
    and electrophoresis of nasal mucus glycoproteins from rats exposed to
    5 or 20 ppm SO2 in air for 7 days showed the appearance of 3 to 5
    new bands in the acidic fraction which were attributed to increased
    cross-linking; the effect was first noticeable after 2 hours exposure.
    The polymerization of glycoprotein molecules may account for the
    decrease in the nasal mucus flow rate and increased viscosity commonly
    associated with inhalation of sulfur dioxide (Gause & Barker, 1978).

         It was found that sulfitolysis of seven disulfide bonds in bovine
    serum albumin caused topographical changes resulting in an 80%
    reduction in its ability to bind to antiserum (Habeeb, 1971).

    Effects on thiamine

         It has been known for many years that treatment of foods with
    sulfites reduces 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 (Hötzel, 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 was 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. 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 SO2, 50% of the thiamine was
    destroyed in one week. However, no loss of thiamine was observed in 48
    hours. The small amount 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,

         A group of subjects on a thiamine-deficient diet were given
    400 mg sulfite/person/day. The diet produced signs of vitamin
    deficiency in 50 days. In another experiment, 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, or by determination of thiamine-dependent
    enzyme activity.  Clinical, neurophysiological, and biochemical
    investigations produced no indication of adverse effects from sulfite
    (Hötzel et al., 1969).

         Other work supports the view that SO2 in beverages does not
    reduce the level of thiamine in the rest of the diet (Sharratt, 1970).

         It has not been demonstrated that destruction of thiamine by
    sulfite in vivo is sufficient to deplete reserves of thiamine nor
    that the symptoms of bisulfite toxicity are coincident with thiamin
    deficiency (Gunnison et al., 1981a).

    Effects on cyanocobalamin

         Rats fed a diet containing 6% sodium metabisulfite for 21 days
    became severely anaemic; destruction of cyanocobalamin by high
    concentrations of sulfite in the diet or gut was considered a possible
    mechanism in the production of anaemia (Gunnison et al., 1981a).

         Cyanocobalamin has been claimed to be an effective blocking agent
    for sulfite-induced bronchoconstriction in asthmatics, but the
    mechanism is unexplained (Jacobsen et al., 1984).

    Effects on enzymes

         Sulfite is a strong inhibitor of some dehydrogenases, e.g.
    lactate dehydrogenase (heart) and malate dehydrogenase; 50% inhibition
    was caused by about 10-5 M sulfite (Pfleiderer et al., 1956).

         Flavins in the flavoproteins form chemical adducts with sulfites
    leading to sulfonation at the N5 atom of flay in, the active site
    that accepts hydrogen. This is the probable mechanism of inhibition of
    dehydrogenases. Several flavoprotein oxidases (e.g. D-and L-amino
    oxidases, oxynitrilase, lactate oxidase, and glycolate oxidase) form
    stable adducts, with dissociation constants from 10-3 to 10-7 M.
    The flavoprotein dehydrogenases did not form adducts with sulfite at
    concentrations of 20 mM (Muller & Massey, 1971).

         Cytochrome oxidase was inhibited 37% by 0.5 mM sulfite at pH 7
    (Cooperstein, 1963), and alpha-glucan phosphorylase was inhibited by
    10 to 30 mM sulfite (Kamogawa & Fukui, 1973). Sulfite was a
    competitive inhibitor of phosphate in glycogen synthesis and
    degradation, and alkaline phosphatase was inhibited in vivo.
    Conversely, the activity of 2,3-diphosphoglyceric acid phosphatase was
    enhanced 15-fold by 2.5 mM sulfite (Harkness & Roth, 1969). Sulfite
    has been found to be a potent inhibitor of many sulfatases
    (Roy, 1976).

    Effects on calcium balance

         Interest in this aspect arises from the possibility that sulfate
    formed metabolically from sulfite may serve to increase the loss of
    calcium in urine and faeces of man.

         Levels of 0.5 to 0.7% calcium carbonate in the diet caused
    increased faecal excretion and diminished urinary levels of calcium.
    Levels up to 0.2% had no effect on the excretion of calcium (Causeret
    & Hugot, 1960).

         Diets containing 0.5 and 1% calcium carbonate and 0.5 and 1%
    potassium metabisulfite (0.29 and 0.58% as SO2 respectively) were
    administered to young rats, and the faecal and urinary excretion of
    calcium were measured for 10 days. At the lower level of dietary
    calcium, both levels of the metabisulfite caused a significant
    increase in the urinary excretion of calcium but had no effect on the
    faecal excretion of calcium. At the higher dietary calcium level, 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).

    Effects on vitamin A

         The levels of hepatic vitamin A were determined in both control
    and test rats receiving 1.2 g/l potassium metabisulfite in the
    drinking water (700 mg/l as SO2). There was a slight decrease in the
    vitamin A level in the liver of test animals after 10 days. In another
    experiment, two groups of 40 rats were kept for four months on a diet
    containing only traces of vitamin A. The drinking water of one group
    contained 1.2 g/l 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).

    Effects on lipids

         Low concentrations of bisulfite (0.5 mM) induced oxidation of
    corn oil emulsified in 1.5% polysorbate solution (Kaplan et al.,
    1975), and similar effects were reported in liver homogenates
    (Inouye et al., 1978).

         Incubation of unsaturated membrane lipids with a large excess of
    bisulfite caused changes in the chromatographic behaviour indicative
    of addition of bisulfite across double bonds. Such changes in membrane
    lipids could account for the irritant effect of sulfur dioxide
    (Akogyeram & Southerland, 1980).

         In brains of guinea-pigs exposed to 10 ppm sulfur dioxide in air
    for 1 hour daily for 21 days, total lipids and free fatty acids were
    decreased in all regions of the brain, but changes in other fractions
    varied with the region. The rates of peroxidation and the activity of
    lipase were increased significantly in all regions of the brain
    (Haider et al., 1981).

    Toxicological studies

    Special studies on carcinogenicity

    (see also Long-term studies)


         Groups of 50 male and 50 female ICR/JCL mice received potassium
    metabisulfite in drinking water at concentrations of 0.1 or 2% for 24
    months. At termination detailed pathological examination did not
    reveal any increase in tumour incidence in treated animals relative to
    controls (Tanaka et al., 1979).


         In a study not designed as an orthodox carcinogenicity bioassay,
    4 out of 149 female rats with low sulfite oxidase activity induced by
    low molybdenum diets in association with tungstate treatment displayed
    mammary adenocarcinomas after 9 weeks of treatment. The animals were
    not exposed to exogenous sulfite and no tumours were seen in control
    animals with normal levels of sulfite oxidase (Gunnison et al.,

    Special studies on mutagenicity

         Using E. coli as an indicator, the frequency of mutation of the
    C gene of phage-lambda was shown to be increased by a factor of 10,
    when compared with controls, by treatment with 3 M sodium hydrogen
    sulfite at pH 5.6 at 37°C for 1.5 hours (Hayatsu & Miura, 1970).
    Sodium hydrogen sulfite induces mutations in only those mutants which
    have cytosine-guanine at the mutant site (Mukai et al., 1970).

         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 this finding 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 SO2 for periods of 8 hours, provided a recovery
    period followed each exposure. In another study at higher
    concentrations of SO2, growth was comparable to that in control
    cultures at 500 ppm SO2, while there was inhibition of growth at
    2000 ppm SO2. The addition of salts of SO2 caused stimulation of
    growth at lower levels, and complete inhibition at 2000 ppm sodium
    hydrogen sulfite (Thompson & Pace, 1962).

         Bisulfite at a concentration of 10 mM (pH not specified) induced
    mutations in Staphylococcus aureus; 5 mM bisulfite induced mutations
    in Saccharomyces cerevisiae at pH 3.6, but not at pH 5.5 (Shapiro,
    1983). Bisulfite at a concentration of 0.1 M was non-mutagenic to
    E. coli (Mallon & Rossman, 1981). At concentrations similar to those
    found in wine (150 ppm, pH 3.0-6.5), bisulfite was not mutagenic to
    Bacillus subtilis (Khoudokormoff, 1978).  Higher concentrations of a
    sodium sulfite-bisulfite mixture showed mutagenic effects in a
    B. subtilis test system at concentrations of 0.1 to 0.5 M, pH 7, but
    not at 0.05 M. Cells treated with adductS of sodium hydrogen sulfite
    and cytidine monophosphate or uridine monophosphate exhibited
    mutagenic effects at concentrations of 0.25 and 0.5 M (Chang et al.,

         Sulfite forms reversible adducts with the 5,6-double bond of
    cytosine and uracil and their nucleosides and nucleotides. The
    reaction is pH- and concentration-dependent (Gunnison, 1981; Shapiro,
    1983). Deamination of cytosine to uracil nucleotides in single-
    stranded DNA occurs in bisulfite solutions of 1 M or higher at pH 5-6
    (Bayatsu, 1976; Shapiro, 1983). The cytosine adduct can be
    transaminated with primary and secondary amines, including lysine,
    with the formation of N4-substituted cytosines.  Cross-linking of
    heat-denatured calf thymus DNA occurred after 6 days in 0.15 M sodium
    hydrogen sulfite, but double-stranded DNA did not cross-link (Shapiro
    & Gazit, 1977).

         In cultures of mouse hepatocytes, HeLa cells, human embryonic
    lung cells, lymphocytes, and oocytes, inhibition of DNA synthesis was
    observed at bisulfite concentrations from 0.1 to 10 mM (Shapiro,
    1983). Intranucleotide bonds of DNA were cleaved by 1 to 10 mM sodium
    hydrogen sulfite solutions by a mechanism believed to involve free
    radical formation (Hayatsu & Miller, 1972).

         Transformation of Syrian hamster embryo cells occurred in a
    dose-dependent manner on treatment with 1, 5, or 10 mM bisulfite for
    24 hours, but the authors suggested that this might not occur by a
    mutagenic mechanism (DiPaolo et al., 1981). Further work in this
    system indicated that bisulfite caused no detectable DNA damage and
    may have decreased the rate of DNA replication by blocking the
    operation of part of the functioning replicons (Doniger et al.,

         Dose- and time-dependent induction of sister chromatid exchange
    was demonstrated in Chinese hamster ovary cells following exposure to
    0.03 to 7.3 mM bisulfite for 2-24 hours (MacRae & Stich, 1979). In
    contrast, Chinese hamster V-79 cells exhibited no mutations to ouabain
    resistance after exposure to 10 and 20 mM bisulfite for 15 minutes.
    Similarly, exposure to 10 mM bisulfite produced no mutations to
    6-thioguanine resistance. Long-term exposure of V-79 cells (recultured
    for 8 weeks in a medium containing 5 mM bisulfite) failed to induce
    ouabain-resistant mutations (Mallon & Rossman, 1981).

         Cultures of human lymphocytes exhibited chromosomal abnormalities
    (clumping), decreased DNA synthesis, cell growth, and mitotic indices
    after exposure to 100 ml of 5.7 ppm sulfur dioxide in air on days 0 or
    1 of incubation but not on days 2 or 3 (Schneider & Calkins, 1970).

         Mutagenic effects have not been reported in whole animals exposed
    to sulfur dioxide or sulfites. Dominant lethal mutations were not
    observed in female C3Hx101 mice given a single i.p. injection of
    550 mg sodium hydrogen sulfite/kg b.w. and mated with untreated males.
    In the same study, neither heritable translocations nor dominant
    lethal mutations occurred when male mice were mated after receiving
    i.p. injections of 400 mg sodium hydrogen sulfite/kg b.w. 20 times
    over a 26-day period or 300 mg/kg b.w. 38 times over a 54-day period
    (Generoso et al., 1978).

         Chromosomal aberrations were not found in oocytes of female Camm
    mice given i.v. doses of 1.0, 2.5, or 5.0 mg sodium sulfite, although
    structural abnormalities were reported when cultures of Camm mouse
    oocytes were treated with sodium sulfite in vitro (Jagiello et al.,

         The influence of low levels of sulfite oxidase activity on
    cytogenetic effects was studied in Chinese hamsters and NMRI mice made
    sulfite oxidase-deficient by treatment with tungstate in association
    with low molybdenum diets. No effects on sister chromatid exchange,
    chromosomal aberrations, or micronucleus tests were seen in either
    species after oral, s.c., or i.p. administration of sodium
    metabisulfite, although control animals tolerated higher doses of
    metabisulfite than those made sulfite oxidase-deficient (Renner &
    Wever, 1983).

         Synergistic effects of sulfur dioxide and sulfites with other
    treatments have been studied for possible co-mutagenic effects.
    Mutation frequency was approximately doubled in UV-irradiated Chinese
    hamster V-79 cells exposed to 10 mM bisulfite either during or after
    irradiation. Tryptophan revertants were increased more than eight-fold
    in U.V.-irradiated E. coli cells exposed to 75 mM bisulfite
    (Mallon & Rossman, 1981). Treatment of phage-lambda with bisulfite and
    several amines (1 M bisulfite plus 1 M semicarbazide, hydrazine,
    methoxyamine, or hydroxylamine) caused an increase in mutation
    frequency (plaque-forming activity) compared with treatment with
    bisulfite alone (Hayatsu & Kitajo, 1977). Combinations of bisulfite
    (150 ppm) with nitrite (100, 200, or 400 ppm) were reported to be
    weakly mutagenic in B. subtilis (Khoudokormoff, 1978). Mutagenic
    effects of coffee on S. typhimurium strains TA98 and TA100 without
    S-9 preparations were completely inhibited by the addition of 300 ppm
    sulfite, bisulfite, or metabisulfite, and the activity of coffee in
    the prophage-lambda induction test was also suppressed (Suwa et al.,
    1982). Sodium sulfite was a weak inhibitor of benz(a)pyrene
    mutagenicity in S. typhimurium TA98 (Calle & Sullivan, 1982).
    Bisulfite concentrations of 0.5, 2.5, and 5.0 µg/ml and the much
    higher concentration of 100 mg/ml inhibited transformation of C3H 1OT
    1/2 cells by X-rays or benz(a)pyrene; pre-treatment of hamster embryo
    cells with 100 ppm bisulfite inhibited transformation by X-rays
    (personal communication with attachments from C. Borek, Columbia
    University, New York, NY, USA, to S.A. Anderson, Federation of
    American Societies for Experimental Biology (FASEB), Bethesda, MD,
    USA, 1984, submitted to WHO by FASEB).

    Special study on reproduction


         Six groups of 20 male and female rats were mated after 21 weeks
    on diets containing 0, 0.125, 0.25, 0.5, 1.0, or 2.0% sodium
    metabisulfite; 10 males and 10 females were remated at 34 weeks. Ten
    male and 10 female F1a rats were mated at 12 and 30 weeks of age to
    give F2a and F2b offspring. Ten males and 15 females of the F2a
    generation were then mated at 14 and 22 weeks to give F3a and F3b
    offspring. F1a parents and F2a parents were kept on the diet for
    104 and 30 weeks, respectively.

         Pregnancy incidence, birth weight, and postnatal survival were
    all normal. In the F0 first mating, the body-weight gain of
    offspring was decreased at 2% sodium metabisulfite and in the F1
    mating it was decreased at 1 and 2% sodium metabisulfate. 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%
    sodium metabisulfate and above in the first F2 mating. The body
    weights of F0 adults were unaffected, while high-dose F1 females
    and high-dose F2 males and females both showed slightly decreased
    body-weight gains (Til et al., 1972a).

    Special study on teratogenicity

         Reproductive performance was studied in normal female
    Wistar-derived rats and in similar rats treated with tungstate to
    induce sulfite oxidase deficiency. The rats received 25 or 50 mM
    sodium metabisulfite in drinking water from 3 weeks prior to mating
    until day 20 of gestation. No treatment-related effects on
    reproductive performance or incidence of malformations were observed
    (Dulak et al., 1984).

    Acute toxicity
                                              LD50 (mg/kg b.w.)
    Species     Route     Hydrogen sulfite    Sodium sulfite     Reference

    Mouse       i.p.      675                 -                  Wilkins et al., 1968
                i.v.      130                 175                Boppe & Goble, 1951

    Rat         i.p.      500-740             -                  Wilkins et al., 1968
                i.v.      115                 -                  Hoppe & Goble, 1951

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

    Rabbit      oral      -                   600-700            Rost & Franz, 1913
                                              (as SO2)
                i.p.      300                 -                  Wilkins et al., 1968
                i.v.      65                  -                  Hoppe & Goble, 1951

    Dog         i.p.      244                 -                  Wilkins et al., 1965
    Short-term studies


         In thiamine-deficient rats, daily oral administration of fruit
    syrup containing 350 ppm sulfur dioxide at 0.5 ml/150 g b.w. for 8
    weeks failed to influence growth (Lockett, 1957).

         Groups of weanling rats numbering 5 per group were fed 0.6%
    sodium metabisulfite (not less than 0.34% as SO2) for 6 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 & Lockett, 1964).

         Three groups of 20 to 30 rats containing equal numbers of males
    and females received daily doses of sulfite dissolved in water or
    added to wine; a control group received the same volume of water. The
    levels of sulfite in the two groups receiving wine were equivalent to
    105 or 450 mg SO2 per litre, and the aqueous solution contained
    potassium metabisulfite equivalent to 450 mg SO2 per litre. The
    effect of this treatment was studied in 4 successive generations, the
    duration being 4 months in females and 6 months in males. Groups of
    animals from the second generation were treated for 1 year. No effects
    were observed on weight gain, efficiency of utilization of protein,
    biological value of the same protein, or reproduction. There was 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 (Personal
    communication of work in progress from P. Jaulmes, 1964).

         Rats were fed sulfite as sodium metabisulfite in stock or
    purified diet at levels from 0.125 to 6% for up to 8 weeks. In a
    preliminary study, increasing levels of sulfite (0.125 to 2.0%
    in the diet) resulted in decreased urinary thiamine excretion.
    Supplementation of the diet with 50 mg thiamine/kg diet 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, 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%). Histopatho-
    logical changes in the stomach occurred at 1% metabisulfite and above
    (Til, 1970).

         Groups of 10 male and 10 female rats were fed diets containing 0
    to 8% sodium metabisulfite for 10-56 days. Vitamin deficiency was
    prevented by adding thiamine to the diet. Diets containing 6% and
    above metabisulfite depressed food intake and growth; glandular
    hyperplasia, haemorrhage, ulceration, necrosis, and inflammation of
    the stomach occurred. Anaemia occurred in all animals receiving 2%
    metabisulfite and above and a leucocytosis occurred in those receiving
    6% metabisulfite. At 4% and above, splenic haematopoiesis was found. 
    The effects were reversible when metabisulfite was removed from the
    diet (Til, 1970).

         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 a
    control. No effects were observed after treatment for 3 months on
    reproduction, mortality, or blood count. The second and third
    generations were treated in the same way for 3 months, the only effect
    observed being 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 dietary regime
    (personal communication from J. Causeret to WHO, 1964).

         Groups of 20 Wistar rats (10 of each sex) were fed diets
    containing 0.125, 0.25, 0.5, 1.0, or 2.0% sodium hydrogen sulfite
    (0.077-1.23% 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
    respective 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; the control and
    2.0% female groups, which were used for fertility studies, gave birth
    to litters during the course of the test and 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 7-8 weeks (all
    groups) and at 13 weeks (2% group and controls) revealed no effect of

         Thiamine could not be measured in the diet containing 2% sulfite
    after being stored for 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 were 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 firm conclusions to be drawn.

         Males and females of the control and 2% groups were mated with
    rats drawn from the main colony. The only adverse findings observed in
    females of the 2% group were lower weight of the offspring at 7 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 was claimed that
    no changes were found in relative organ weight (liver, heart, spleen,
    kidneys, adrenalin, and testes) nor in microscopic appearance (above
    organs, and the stomach, intestine, uterus, teeth, and eyes)
    (CIVO/TNO, 1965).

         In a study of the gastric lesions produced in short-term studies
    at high dose levels of metabisulfite, Cpb:Wu Wistar rats were fed
    thiamine-supplemented diets containing 0, 4, or 6% sodium
    metabisulfite for 8 or 12 weeks and 0 or 6% sodium metabiaulfite for
    4, 7, 14, 21, or 28 days in a related time-course study. In the
    subchronic study, the fundic mucosa of the treated rats contained
    scattered hyperplastic glands lined with enlarged gastric chief cells
    containing large numbers of pepsinogen granules that were devoid of
    fat, glycogen, or mucus. The time course study suggested that
    pre-existing chief cells were transformed to hyperactive chief cells
    having proliferative capability. The pathogenesis of these lesions
    remains to be clarified (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 on post mortem
    examination. 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 for between 46 and 171 days lost weight, and autopsy
    showed stomach haemorrhages (Rost & Franz, 1913).


         A daily dose of 3 g sodium sulfite was given by stomach tube to a
    dog weighing 17 kg for 23 days. Another dog weighing 34 kg was given
    6-16 g 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
    hydrogen sulfite 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
    1.082.51 g. Examination of heart, lungs, liver, kidneys, 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
    containing 0, 0.06, 0.16, 0.35, 0.83, or 1.72% sodium metabisulfite.
    Fourteen males and 14 females/group were sacrificed at 15-19 weeks and
    the remainder were killed 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% sodium metabisulfite. Food
    intake and weight gain were reduced at the 1.72% level; however, in
    the paired-feeding study, growth and food conversion were not
    affected. Mortality was not related to metabisulfite ingestion.
    Urinary and liver thiamine levels decreased with increasing dose, but
    they were reduced below the levels found in pigs on basal diet alone
    only at 1.72%. Haematology and faecal occult blood determinations were
    comparable in all groups. Organ/body-weight ratios were elevated at
    0.83 and 1.72% for the heart, kidneys, and spleen, and at 1.72% for
    the liver. The paired-feeding study showed liver- and kidney-weight
    ratios to be increased at 1.72% metabisulfite. Mucosal folds in the
    stomach and black colouration of the caecal mucosa at the top 2 dose
    levels were observed on gross pathological examination. At 0.83 and
    1.72% metabisulfite, histopathological examination showed hyperplasia
    of mucosal glands and surface epithelium in the pyloric and cardiac
    regions. Intra-epithelial micro-abscesses, epithelial hyperplasia, and
    accumulations of neutrophilic leucocytes in papillae tips were
    observed in the pars oesophagea. 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% metabisulfite,
    fat-containing Kupffer cells were present in unusually high numbers in
    the liver (Til et al., 1972b).

    Long-term studies


         Groups of rats numbering from 18 to 24 per group were fed sodium
    hydrogen sulfite at dosages of 125, 250, 500, 1000, 2500, 5000,
    10,000, or 20,000 ppm of the diet for periods ranging from 1 to 2
    years. The rats fed 500 ppm sodium hydrogen sulfite (307 ppm as SO2)
    for 2 years showed no toxic symptoms. Sulfite at concentrations of
    1000 ppm (615 ppm 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, 350, or 750 ppm SO2, respectively. Prior interaction of the
    sulfite with dietary constituents was thus prevented. The experiment

    lasted 2.5 years and extended over 3 generations of rats. No effects
    were observed on food consumption, fluid intake, faecal output,
    reproduction, lactation, or the incidence of tumours (Lockett &
    Natoff, 1960).

         A solution containing 1.2 g of potassium metabisulfite per litre
    of water (700 ppm as 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. The intake of fluid by the test
    group was the same as that of the controls (but no measurements of
    SO2 loss from the metabisulfite solution appear to have been made).
    The intake of SO2 calculated from the consumption of water was
    3060 mg/kg b.w./day for males and 40-80 mg/kg b.w./day for females.
    The following observations provided no evidence of toxic effects;
    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
    higher 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
    effects of treatment 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 a standard diet) were
    given daily doses (30 ml/kg b.w.) of red wine containing 100 or
    450 ppm SO2, an aqueous solution of potassium metabisulfite (450 ppm
    SO2), or pure water by oral intubation on 6 days each week for 4
    successive generations. The females were treated for 4 and the males
    for 6 months; the second generation was treated for 1 year. The only
    effect seen was a slight reduction in hepatic cellular respiration.
    All other parameters examined, which comprised weight gain, weight and
    macroscopic or histological appearance of various organs, appearance
    and behaviour, proportion of parturient females, litter size and
    weight, and 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% sodium metabisulfite in a diet enriched with 50 ppm
    thiamine for 2 years. Animals were stressed by breeding at 21 weeks,
    and again by breeding of half of each group at 34 weeks (see Special
    study on reproduction). 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 by treatment. Thiamine content of
    the urine and liver showed a dose-related decrease commencing at 0.125

    and 0.25% metabisulfite, respectively. However, thiamine levels at 2%
    metabisulfite were comparable to thiamine levels in control rats.
    Marginally-reduced haemoglobin levels were noted on 3 occasions in
    females in the 2%-dose group, and occult blood was noted in faeces at
    1% metabisuliite and above. In 10% of the females at 0.25%
    metabisulfite, and in 10% of the males at 0.5% metabisulfite, 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% metabisulfite and above. The
    incidence of neoplasms was not increased above normal levels at any
    site at any dose. The NOEL in this study was 0.25% sodium
    metabisulfite (Til et al., 1972a).

    Observations in man

         In man, a single oral dose of 4 g of sodium sulfite caused toxic
    symptoms in 6 of 7 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
    b.w. (Lafontaine & Goblet, 1955).

    Idiosyncratic sensitivity to sulfites

         The most commonly-reported adverse reaction to sulfur dioxide or
    sulfiting agents in man is bronchoconstriction and bronchospasm,
    particularly among a sensitive sub-group of asthmatics. Less commonly,
    symptoms similar to anaphylaxis, flushing, hypotension, and tingling
    sensations have been reported (NIH, 1984).

         A mildly-asthmatic child was reported to suffer acute, transient
    asthmatic episodes following ingestion of sulfited foods, but no
    controlled challenge test was performed (Kochen, 1973).

         Of 272 asthmatic patients, 30 were reported to experience
    bronchoconstriction after ingesting orange drinks containing sodium
    hydrogen sulfite. Challenge tests on 14 of the 30 sensitive ptients
    using a single dose of 25 mg sodium metabisulfite (100 ppm in an
    acidic solution) resulted in a fall of at least 12% in FEV1 (forced
    excretory volume in one second) in 8 subjects within 2 to 25 minutes.
    No placebo test was performed (Freedman, 1977).

         An asthmatic patient who experienced bronchospasm after ingestion
    of canned crabmeat salad with vinegar dressing developed severe
    bronchospasm within 30 minutes of receiving an oral challenge (dose
    not stated) of sodium metabisulfite; no reaction occurred after

    ingestion of crabmeat alone. A second patient, whose asthma was
    provoked by wine, was given a single-blind oral challenge with a
    capsule containing 500 mg sodium metabisulfite. Peak respiratory flow
    rate fell from 440 1/min. before challenge to 100 1/min. afterward. No
    effect was observed with a lactose placebo (Baker et al., 1981).

         Four patients with histories of severe bronchoconstriction and
    anaphylaxis associated with restaurant meals were subjected to a
    single-blind challenge test. Placebo capsules (lactose) were
    administered at 30 min. intervals to the fasting subjects on the first
    day and capsules containing 1, 5, 10, 25, and 50 mg potassium hydrogen
    sulfite were given sequentially on the following day. All 4 patients
    developed asthmatic symptoms after challenge doses of 10, 25, and
    50 mg. FEV fell 34-49% at 30 to 90 minutes after challenge. Subjective
    symptoms (flushing, tingling, and/or faintness) were also reported. No
    adverse reactions to an oral challenge with bisulfite were reported in
    5 steroid-dependent asthmatics without histories of adverse reactions
    to restaurant meals (Stevenson & Simon, 1981).

         Fifteen asthmatics with histories of asthmatic reactions to food
    and beverages were challenged sequentially with oral capsules
    containing 5, 10, 25, and 50 mg sodium metabisulfite. Only one subject
    reacted significantly to the challenge, with a fall of 28% in FEV
    within 2 minutes of receding a dose of 5 mg sodium metabisulfite
    (Koepke & Selner, 1982).

         Oral challenge of 6 sulfite-sensitive asthmatics with solutions
    of sulfite produced reactions at doses approximately half those
    required to produce similar reactions when given in capsule form
    (Goldfarb & Simon, 1984).

         Administration of atropine, cromolyn, doxepin, or vitamin B12
    prior to sulfite challenge ameliorated the asthmatic reaction of 6
    sulfite-sensitive subjects (Simon et al., 1984).

         Twelve patients with idiopathic anaphylaxis, 9 of whom had a
    history of adverse reactions to restaurant meals, and 10 control
    subjects received sequential challenges with increasing oral doses
    (1, 5, 10, 25, 50, 100, and 200 mg) sodium metabisulfite in lemonade.
    A similar degree of non-specific irritant and subjective symptoms were
    reported in both groups and no anaphylactic reactions were observed.
    Pulmonary function was abnormal in 3 subjects, but no bronchospasm was
    induced (Sonin & Patterson, 1985).

         Sequential capsule challenge tests with similar doses of sodium
    bisulfite in 32 patients (14 with recurrent idiopathic anaphylaxis, 8
    with systemic mastocytosis, and 10 with reported allergic reactions to
    meals) resulted in anaphylactic reactions in 2 of the idiopathic
    anaphylaxis patients, but these 2 patients responded similarly to the
    placebo challenge (Metcalfe, 1984).

         Of 61 asthmatic patients with no history of sulfite sensitivity,
    5 patients (8.2%) had a fall in FEV1 of more than 25% following a
    single-blind oral challenge with increasing capsule doses (10, 25, 50,
    and 100 mg) of bisulfite at 30-minute intervals. Patients who did not
    react to this challenge were further challenged with acidic solutions
    of 1 or 10 mg of sodium metabisulfite. The symptoms reported in the 5
    patients who reacted to the challenge were milder and required larger
    challenge doses than in sulfite-sensitive asthmatic patients with a
    history of reactions to restaurant meals (Simon et al., 1982).

         Pulmonary function was assessed in 25 asthmatic and 25
    non-asthmatic subjects before and after consuming 112 ml wine
    containing 140 mg sulfur dioxide per litre. A decrease of more than
    12% in FEV1 occurred in 1 non-asthmatic and 5 asthmatic subjects.
    Two of the asthmatic responders were challenged with 2 solutions, one
    a model solution containing all the wine ingredients except sulfur
    dioxide and the other a metabisulfite solution alone. Both subjects
    displayed a fall in FEV1 after receiving metabisulfite alone and one
    also reacted to the model wine solution without metabisulfite
    (Seyal et al., 1984).

         Five sulfite-sensitive asthmatic patients with a history of
    adverse reactions to sulfited foods were challenged with lettuce
    treated with a commercial vegetable freshener containing sodium
    hydrogen sulfite; a control experiment was performed using lettuce
    treated with a commercial freshener not containing sulfite.
    Approximately 10 ml of the freshener solution (80-90 mg sodium
    hydrogen sulfite) adhered to the 3-ounce portions of lettuce used. All
    the patients displayed a reduced FEV1 (mean decrease 44%, range
    31-64%) after the challenge with sulfited lettuce but not after
    receiving the control lettuce. Four of the patients were described as
    having moderate asthmatic reactions, while the fifth reacted severely
    and required extensive emergency treatment (Howland & Simon, 1985;
    Simon, 1984).

         Two patients who showed symptoms (dizziness, weakness, nausea,
    chest tightness, tachycardia, and dyspnea) associated with restaurant
    meals reported vague, general symptoms after an oral challenge with
    sodium metabisulfite, but pulmonary function showed no changes after
    challenge (Schwartz, 1953).

         Estimates of the frequency of sulfite sensitivity among
    asthmatics have been made, based on experimental studies, but they are
    complicated by the fact that different end-points have been used to
    define an adverse reaction and by bias in the sample population
    (e.g. in referral practices where a disproportionate number of
    severely affected, steroid-dependent asthmatic patients are seen).

         Capsules containing 1.4, 14, 144, and 288 mg potassium
    metabisulfite were given sequentially to 134 asthmatic patients
    selected from a clinic population of 1073 patients with asthma and
    related symptoms. Decreases in FEV1 of more than 15% were reported
    in 50 of the subjects challenged. Based on this study, it was
    estimated that 4.6% of asthmatic patients respond to sulfite challenge
    (Buckley et al., 1985). Oral challenge studies with potassium
    metabisulfite on 100 non-steroid-dependent asthmatic subjects resulted
    in no cases of sulfite sensitivity that could be confirmed in
    double-blind trials. Single-blind challenge studies on 69 steroid-
    dependent asthmatics resulted in a greater than 20% decrease
    in FEV1 in 14 subjects. Double-blind challenges of 5 of these
    patients resulted in a significantly-decreased FEV1 in 2 cases.
    Based on these studies, it was estimated that 5-10% of steroid-
    dependent asthmatics and 1-2% of all asthmatics may be sulfite
    sensitive (Taylor, 1984). Other workers have suggested that 5-10% of
    asthmatics may be sulfite sensitive (Simon et al., 1982; Simon,
    1984). However, Patterson (personal communication with attachments
    from R. Patterson, Northwestern University, Evanston, IL, USA, to S.A.
    Anderson, Federation of American Societies for Experimental Biology
    (FASEB), Bethesda, MD, USA, 1984, submitted to WHO by FASEB) failed to
    identify sulfite sensitivity among idiopathic anaphylactic patients
    from an extensive population of asthmatics, from which he concludes
    that sulfite sensitivity may be a minor problem.

         Bronchoconstriction and bronchospasm occur with greater frequency
    after inhalation of sulfur dioxide than after oral ingestion of
    sulfites in both asthmatic and non-asthmatic individuals (Koenig
    et al., 1982; Nadel et al., 1965; Schachter et al., 1984;
    Sheppard et al., 1980). Inhalation of bronchodilators containing
    sulfites has also been associated with bronchospasm and anaphylaxis
    (Koepke et al., 1984; Twarog & Leung, 1982). Inhalation of sulfur
    dioxide by 6 known sulfite-sensitive asthmatics induced falls in FEV1
    of more than 25% at doses of 1 to 10% of that required by ingestion
    (Goldfarb & Simon, 1984).

         It is possible that inhalation of sulfur dioxide after eructation
    may have occurred in some oral challenge studies. Concentrations of
    4-50 ppm sulfur dioxide were reported in the stomach contents of 5
    subjects challenged with 25 or 50 mg metabisulfite under unspecified
    conditions (Allen & Delohery, 1985). Inhalation of air containing
    sulfur dioxide in the headspace gases of sulfited foods may also be a
    contributory factor. A patient who had a wheezing episode after
    inhalation of headspace gas from sulfited dried apricots did not react
    to an oral challenge of 50 mg sodium metabisulfite (Werth, 1982). It
    was estimated that the airspace above an aqueous solution of 70 ppm
    sulfur dioxide contained 1 ppm sulfur dioxide at room temperature
    (Freedman, 1977), a concentration causing bronchoconstriction in some
    asthmatics (Sheppard et al., 1980). Administration of a mouthwash

    containing up to 100 mg sodium metabisulfite in 30 ml citric acid
    solution resulted in a fall of more than 20% in FEV1 in 9 of 15
    asthmatics, but the 9 who reacted did not respond when they held their
    breath during the challenge (Allen & Delohery, 1985).

         I.v. infusion of sulfite-containing medication (theophylline and
    dexamethaone) seriously worsened an asthmatic attack in a patient who
    had previously shown a large decrease in peak flow rate following an
    oral capsule challenge of 500 mg sodium metabisulfite. I.v. injection
    of metaclopromide, which contained metabisulfite, also caused
    bronchospasm in this patient (Baker et al., 1981). Injection of a
    dose of lidocaine hydrochloride containing metabisulfite was followed
    by plantar pruritis in an individual who had complained of similar
    symptoms after eating chili soups, sandwiches, salads, jalapenos,
    pizza, Chinese pickled green turnips, or dried shrimp (Huang & Fraser,

         Conversely, patients receiving total parenteral nutrition (TPN)
    solutions preserved with bisulfites may receive up to 950 mg bisulfite
    per day, but no sensitivity reactions have been associated with this
    practice (Metcalfe, 1984). One report of excretion of abnormal sulfur
    metabolites in a patient receiving TPN for 18 months (Abumrad et al.,
    1981) led to the suggestion that tachypnoea in this patient might have
    been associated with abnormal metabolism of sulfite (Gunnison &
    Jacobsen, 1983).

         As indicated above, there is normally a considerable reserve of
    activity of sulfite oxidase in man, but a few cases of sulfite oxidase
    deficiency have been identified (Irreverre et al., 1967; Shih et al.,
    1977; Duran et al., 1979; Ogier et al., 1982). In these extreme
    cases, the symptoms included severe neurological injury, dislocated
    ocular lenses, and premature death. Sulfite oxidase activity in
    hepatic tissue and/or skin fibroblasts from these individuals was
    below detectable levels (Johnson & Rajagopalan, 1976a,b; Shih et al.,
    1977; Johnson et al., 1980; Ogier et al., 1982). Sulfite oxidase
    activities in skin fibroblasts of both parents of one of these
    patients were below the lowest normal control level (Shih et al.,
    1977), while in another case both parents and a brother had activities
    below average but within the control range; all these relatives were
    asymptomatic.  There is currently insufficient information to
    associate sulfite oxidase deficiency with adverse reactions to
    sulfited foods.

         Life-threatening reactions and deaths of 4 asthmatic individuals
    have been reported following ingestion of restaurant meals, including
    foods treated with sulfiting agents (Food Chemical News, 1984).
    Following 2 of the deaths, samples of the foods ordered by the
    deceased were analysed for sulfite. Two samples of lettuce contained
    78 and 409 ppm sulfite, and guacamole contained 272 ppm sulfite

    (Riddle, 1983). In one of these cases, analysis in U.S. FDA
    laboratories of shredded potatoes (cottage fries) indicated a sulfite
    concentration of 96 ppm; the results of re-analysis of the same
    product before and after cooking were 615 and 582 ppm sulfite,
    respectively (Spears, 1984).

         In another case, a sulfite-sensitive asthmatic patient lapsed
    into a coma after consuming a meal including cottage-fried potatoes.
    Analysis revealed that samples of the potatoes used for cottage fries
    contained 2240 ppm sulfite before cooking and 2210 ppm after cooking
    (Whetstone, 1984). The patient remained comatose for 3 weeks, and 6
    months after the incident still displayed several motor and
    neurological deficits (Simon, 1984).

         Adverse reactions in non-asthmatic individuals appear to be rare,
    but a case has been reported of anaphylaxis in a non-asthmatic male
    who consumed a meal including salad sprayed with bisulfite. Oral
    challenge with 10 mg sodium hydrogen sulfite produced erythema,
    itching, nausea, warmness, coughing, and bronchoconstriction for about
    1 hour (Prenner & Stevens, 1976). Contact dermatitis in response to
    sulfite has also been reported (Fisher, 1975), as has hypotension
    without respiratory distress (Schwartz, 1983).


         Earlier data were considered by the Committee in light of the
    results of new studies performed since the last review; these include
    studies on the chemistry of sulfur dioxide in foods and reactions
    with nutrients, metabolism, teratogenicity, mutagenicity, and
    carcinogenicity studies, effects on the gastric mucosa, and, in
    particular, adverse reactions (bronchoconstriction and anaphylactic-
    type reactions) in man.

         In reviewing case studies, and challenge tests relating to
    idiosyncratic sensitivity to sulfiting agents, the Committee noted the
    serious, life-threatening nature of the adverse effects in some cases
    and that a number of fatalities associated with sulfite-treated foods
    have been reported. Such adverse reactions appear to occur principally,
    but not exclusively, in a sub-population of sulfite-sensitive
    asthmatics and are associated with consumption of salads treated with
    sulfite preparations; other vegetable products with high residual free
    sulfite levels or sulfite-treated acidic beverages have less-commonly
    been implicated. Many sulfite-treated processed foods contain a
    substantial proportion of the residual sulfur dioxide equivalents in
    bound form, but it is not known whether bound sulfur dioxide
    contributes to adverse reactions.

         While sulfiting agents can interact with DNA and may induce
    mutations in bacteria, in vivo mutagenicity studies in mammals were
    negative, as were long-term carcinogenicity studies on potassium and
    sodium metabisulfite in mice and rats, respectively. Long-term anti
    3-generation studies in rats receiving metabisulfite in the diet with
    added thiamine showed a no-effect level of 0.125% sodium metabisulfite
    (equivalent to 70 mg SO2/kg.b.w./day).

         Sulfur (IV) oxo anions are normal intermediates in the metabolism
    of endogenous sulfur compounds in mammals and are oxidized enzymically
    to sulfate prior to urinary excretion. In general, there is a large
    reserve capacity of sulfite oxidase and the systemic toxicity of
    sulfites is low. This conclusion is supported by clinical experience
    with total parenteral nutrition solutions preserved with sulfiting

         While recognizing the utility and versatility of sulfiting agents
    as food additives, the Committee recommended that the use of suitable
    alternative technology, where it exists, should be encouraged,
    particularly in those applications (e.g. control of enzymic browning
    in fresh salad vegetables) where the use of sulfites may lead to high
    levels of acute exposure and which have most commonly been associated
    with life-threatening adverse reactions. There is concern about uses
    of sulfiting agents in situations where such use may be unexpected by
    the consumer and no indication of their presence is given. The
    Committee reiterated the view of the twenty-seventh meeting (Annex 1,
    reference 62, section 2.4) in respect of intolerance to food additives
    that appropriate labelling was the only feasible means of offering
    protection to susceptible individuals.

         The ADI was retained. However, the Committee recommended that the
    frequency of idiosyncratic adverse reactions and the relative toxic
    effects of free and bound sulfur dioxide should be kept under review.
    Information on the chemical forms of sulfur dioxide in food is also


    Level causing no toxicological effect

         Rat: 0.25% sodium metabisulfite in the diet, equivalent to
    70 mg/kg b.w./day calculated as SO2.

    Estimate of acceptable daily intake for man

         0-0.7 mg/kg b.w. for sulfur dioxide and sulfur dioxide

    Further work or information


    1.   Additional studies to assess the true frequency of sulfite
         sensitivity in asthmatics.

    2.   Studies to elucidate the frequency and magnitude of asymptomatic
         sulfite oxidase deficiency and its role in sulfite intolerance.

    3.   Studies on the ability of the various forms of bound sulfur
         dioxide in foods to elicit adverse reactions in sulfite-sensitive


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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 18)