SULFUR DIOXIDE AND SULFITES
Explanation
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.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
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,
1973).
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).
TOXICOLOGICAL STUDIES
Special studies on carcinogenicity Mouse
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,
1962).
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,
1981).
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
Mouse
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).
Rat
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.,
1968
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
Rat
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,
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 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).
Rabbit
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).
Dog
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).
Pig
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).
OBSERVATIONS IN MAN
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
thiamine).
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).
Comments
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.
EVALUATION
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)