Taylor 1986

SULFITES IN FOODS: USES, ANALYTICAL METHODS,
RESIDUES, FATE, EXPOSURE ASSESSMENT, METABOLISM,

TOXICITY, AND HYPERSENSITIVITY
STEVE L. TAYLOR,* NANCY A. HIGLEY,*t

AND ROBERT K. BUSH$
*Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706

$William S. Middleton Memorial VeteransHospital, Madison, Wisconsin 53705
#Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
11. Uses of and Exposure to Sulfites in Foods 4
A. Description . . ..... 4
B. Natural Occurrence of Sulfites in Foods . . . . . . . . . . . . . . . . . . . . . . . . 6
C. History of Use of Sulfiting Agents as Food Ingredients 7
D. Current Food Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
E. Methods for Measurement of Sulfite Residue Levels . . . . . . . . . . . . . . 17
F. Chemistry of Sulfites and Fate in Foods . . . 21
G. Treatment Levels versus Residual Levels . . . . . . . . . . . . . . . . . . . . . . . 30
H. Exposure Assessments 30
111. Safety of Sulfites in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
A. Metabolism of Sulfites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
B. Human Challenge Trials 38
C. Animal and Cellular Toxi 39
D. Hypersensitivity to Ingested Sulfites . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
IV. Possible Substitutes and Their Limitations 61
A. Control of Enzymatic Browning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
B. Control of Nonenzymatic Browning . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
C. Use as Antioxidants or Reducing Agents 62
D. Use as an Antimicrobial Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
E. Use as a Bleaching Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
V. Future Research Needs . . . . 63
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
?Resent address: International Flavors & Fragrances, Research & Development, Union Beach,
New Jersey 07735.
1
Copyright 6 1986 by Academic Press, Inc.
All rights of reproduction in any form rewrved.
2 STEVE L. TAYLOR ET AL.
I. INTRODUCTION
Sulfiting agents have a long history of use as food ingredients. The term
sulfiting agents refers to sulfur dioxide (SO,) and several forms of inorganic
sulfite that liberate SO, under the conditions of use. In addition, naturally occur-
ring sulfites are present in many foods. The yeast cultures used in the fermenta-
tion of wines and beers naturally produce a portion of the sulfites found in these
products.
Sulfiting agents are added to foods for many important technical purposes,
including the control of enzymatic and nonenzymatic browning, antimicrobial
action, antioxidant and reducing agent uses, bleaching agent uses, and a variety
of processing aid uses. In many products, the sulfites serve more than one
purpose. Alternatives to the sulfiting agents are not easy to identify. Possible
alternatives usually provide a narrower range of benefits, are often less effective,
and are almost always more expensive.
Sulfiting agents are currently used in a wide variety of food products. Data on
the treatment levels for the sulfites and residual sulfites are not available for some
food products, and wide variations in treatment modes and levels for particular
products are known to occur in the industry.
The analysis of sulfite residues in foods is confused by the rapid reaction
between sulfiting agents and various food components. Sulfites react readily with
reducing sugars, carbonyls, and proteins to yield a variety of organic combined
sulfites. Analytical procedures are available for “free” SO, (SO, and the vari-
ous inorganic sulfite salts) and “total” SO, (free SO, plus some of the combined
forms of sulfite). Processing, storage, and preparation act to lower the available
residual levels of sulfites in foods. The actual levels of free and total SO, in a
particular food product are dictated by the extent of absorption of the sulfites
during treatment, the nature of the processing treatment following sulfite addi-
tion, and the conditions of storage. The actual levels of free and total SO,
remaining in foods at the point of consumption have received less attention. The
fate of sulfites added to specific foods is a largely unexplored area of study. The
rapid reaction of sulfites with food components would be expected to leave little
free SO, in the product at the point of consumption (Green, 1976; Joslyn and
Braverman, 1954; Schroeter, 1966).
Recently, the safety of the continued use of sulfites in foods has been ques-
tioned on the basis of their alleged role in the initiation of asthmatic reactions in
certain sensitive individuals. Numerous cases of sulfite-induced asthma have
been reported in the medical literature since 1977 (Baker etal., 1981; Buckley et
al., 1985; Bush et al., 1986; Stevenson and Simon, 1981b; Towns and Mellis,
1984; Twarog and Leung, 1982) and additional anecdotal reports have been
made to the Food and Drug Administration. These cases of sulfite-induced
SULFITES IN FOODS 3
asthma were confirmed by positive challenges with capsules or solutions contain-

ing inorganic sulfites. Only a small subgroup of the asthmatic population has
sensitivity to sulfites in capsules.
Important questions remain regarding the possibility that sulfited foods might
initiate asthmatic reactions in these sensitive individuals. Although some of the
described patients report asthmatic reactions to sulfited foods and many of the
anecdotal cases involve suspicions of reactions to sulfited foods, only a few
controlled challenges with sulfited foods have been performed with sensitive
asthmatics (Howland and Simon, 1985; Seyal et af., 1984).
We speculate that the degree of hazard posed to sulfite-sensitive asthmatics by
sulfited foods may be considerably diminished by the reactions of the sulfites
with food components. The majority of the challenges described in the medical
literature thus far have involved inorganic sulfites in capsules or in acidic solu-
tions. There can be little doubt that some asthmatics are sensitive to free in-
organic sulfites, although this may be related to the conversion of inorganic
sulfite salts to SO, at acidic pHs. However, as mentioned, most sulfited foods
contain little free inorganic sulfite; sulfited lettuce is an exception (Taylor er af.,
1985). These free inorganic sulfite residues would probably induce reactions in a
manner similar to sulfites in capsules. The combination of sulfites with food
components would drastically lower the free SO, content of most foods, thereby
limiting exposure to these free forms of sulfites. Further research is needed to
determine the effects of ingestion of combined forms of sulfites on the sulfite-
sensitive asthmatics.
Concerns have also arisen in recent years regarding the possibility that many
consumers may be exceeding the Acceptable Daily Intake (ADI) for sulfites,
although the recent report of the Ad Hoc Review Group on the Reexamination of
the GRAS (Generally Recognized as Safe) Status of Sulfiting Agents indicates
that these concerns are probably unwarranted (Life Sciences Research Office,
1985). They estimate that total intake of sulfites as SO, is about 10 mg/cap-
ita/day, which is well below the AD1 of 42 mg for a 60-kg person (Life Sciences
Research Office, 1985). Again, the same questions arise about the relative con-
tributions of free and combined sulfites to the total sulfite intake. The intake of
combined sulfite likely exceeds the intake of free sulfite by many fold. Because
of their stabilities, the combined forms of sulfites would likely pose a lower
hazard to consumers than free sulfites. More research will be required to firmly
establish the relative toxicity of the free and combined sulfites.
In this article, the current uses of sulfites in foods will be examined. The
critical issue of exposure assessment will be explored in a review of the fate of
sulfites in foods, residual levels, and analytical methodology. The questions
about the safety of the use of sulfites in foods will be tackled by reviewing the
available information on the metabolism of free and combined sulfites, the
toxicity of free and combined sulfites, and the hypersensitivity reactions among
certain asthmatics. In the final section of this review, the remaining unresolved
issues will be highlighted with a discussion of future research needs. In our
opinion, further research is necessary before decisions can be made on the future
regulatory status of sulfites, although tremendous pressure is being exerted on
the Food and Drug Administration to make a decision on the status of sulfites.
More information is needed on the responses of sulfite-sensitive asthmatics to
sulfited foods, the comparative reactivities of asthmatics to free and combined
sulfites, the comparative toxicities of free and combined sulfites, the fate of
sulfites in a variety of foods, and the extent of consumer exposure to free and
combined sulfites.
II. USES OF AND EXPOSURE TO SULFITES IN FOODS
A. DESCRIPTION
Sulfur dioxide and several forms of inorganic sulfites that liberate sulfur
dioxide under the conditions of use are food additives known collectively as
sulfiting agents. Sulfur dioxide (SO,), potassium bisulfite (KHSO,), potassium
metabisulfite (K,S,O,), sodium bisulfite (NaHSO,), sodium metabisulfite
(Na,S,O,), and sodium sulfite (Na,SO,) are listed in the Code of Federal Reg-
ulations (CFR) as GRAS provided that they are not used in meats or other foods
recognized as a source of thiamine. However, the GRAS status of these sulfiting
agents is currently being reexamined, and changes may be made (Life Sciences
Research Office, 1985). In addition, other sections of the CFR specifically allow
the use of sulfiting agents in a variety of food-related processes. A list of the CFR
sections and the processes covered by each section is provided in Table I. Note
that all of the GRAS sulfiting agents are presently allowed for use for certain of
these purposes. Potassium sulfite (K,SO,) and sulfurous acid (H,SO,), which
are not GRAS substances, are specifically allowed for use only in the processing
of caramel. Sulfiting agents are also permitted for use in wine and beer, although
the Bureau of Alcohol, Tobacco, and Firearms (BATF) has proposed that the use
of sulfites in wine and beer be curtailed to some extent (Anonymous, 1984).
Presently, the levels of use of the sulfiting agents in most foods are not strictly
limited by regulation. Exceptions are glucose syrup, dextrose monohydrate, and
wine where the maximum allowable residual levels of SO, are specified, and
food starch bleaching where the treatment level of SO, is controlled to a max-
imum of 0.05%. It should be noted that the levels of sulfites used in some
products such as wines are self-limiting because of organoleptic considerations.
The theoretical yield of sulfur dioxide varies for the different forms of the
SULFITES IN FOODS 5
TABLE I
CODE O F FEDERAL REGULATIONS SECTIONS PERTAINING TO THE USE OF SULFlTlNG AGENTS
IN FOODS AND/OR FOOD INGREDIENTS
CFR section Subject Sulfiting agents allowed
2lCFR 182.3616 GRAS status KHSO3

2lCFR 182.3657 GRAS status K2S205
2lCFR 182.3739 GRAS status NaHS03
2lCFR 182.3766 GRAS status Na2S205
2lCFR 182.3798 GRAS status Na2S03
2lCFR 182.3862 GRAS status so2
2lCFR 73.85(2) Caramel H2SO3, Na2SO3. K2SO3
2lCFR 168.111 Dextrose monohydrate SO2 (20 ppm maximum residual)
2lCFR 168.120 Glucose syrup SO2 (40 ppm maximum residual)
2lCFR 172.892 Food starch bleaching agents SO2 (0.05% maximum)
2 ICFR 173.31O(c) Boiler water additives Na2S205,Na2S03
2lCFR 177.1200(c) Cellophane NaHS03, Na2S03
2lCFR 177.1400 Water-soluble, hydroxyethyl All GRAS sulfiting agents
cellulose film
sulfiting agents, as outlined in Table 11. Consequently, different treatment levels

are required with the various sulfiting agents to yield equivalent doses of the
active agent, SO,. For comparative purposes, it is helpful to calculate treatment
levels on the basis of percentage of theoretical yield of SO,. However, it must be
realized that these theoretical yields would almost never be achieved in food
applications because the sulfiting agents react rapidly with food components, can
be volatilized into the atmosphere, or can oxidize to sulfate. As will be empha-
sized later, these reactions are dependent on a number of variables, including
pH, temperature, and storage time.
TABLE I1
THEORETICAL YIELD AND SOLUBILITY O F GRAS SULFITING AGENTS"
~~~~~~ ~
Theoretical yield Approximate solubility

Chemical Formula of so2 (%) @/lo0 ml H20)
Sulfur dioxide so2 100.00 11 at 20°C

Sodium sulfite, anhydrous Na2S03 50.82 28 at 40°C
Sodium sulfite, heptahydrate Na2S03 . 7H20 25.41 24 at 25°C
Sodium bisulfite NaHS03 61.56 300 at 20°C
Sodium metabisulfite Na2S205 67.39 54 at 20°C
Potassium metabisulfite K2S205 57.60 25 at 0°C
Potassium bisulfite KHSO3 53.32 -
Z, From Green (1976).

The Food Chemicals Codex supplies specifications for the food grades of four
of the sulfiting agents. In general, food grade sulfiting agents must be at least
90% pure to meet these standards. Some problems arise in the definition of
sodium bisulfite, since there is some doubt about the existence of sodium
bisulfite in the solid state. It may exist entirely as sodium metabisulfite or as a
mixture of bisulfite and metabisulfite (Green, 1976). For that reason, the Food
Chemicals Codex defines the purity of sodium bisulfite on the basis of SO,
equivalents.
B. NATURAL OCCURRENCE OF SULFITES IN FOODS
In addition to their use as food additives, it must be remembered that the

sulfites can also occur naturally in foods. Foods contain a variety of sulfur-
containing compounds, including the sulfur amino acids, sulfates, sulfites, and
sulfides. These sulfur-containing compounds are interconvertible in some food
systems that possess the appropriate enzymes.
The natural occurrence of sulfites in foods has been most thoroughly studied in
alcoholic beverages such as wine and beer (Eschenbruch, 1974). The ability of
yeasts to produce sulfite has been known since the end of the last century. Sulfite
arises from sulfate via a multienzyme pathway. The sulfite can be converted into
methionine and cysteine, but sulfite always exists in the fermentation medium.
Sulfite can also be converted into H,S and other sulfides, which are organolep-
tically undesirable in wines and beers. Most strains of Saccharomyces cerevisiae
generate between 10 and 30 ppm SO,, although some strains producing in excess
of 100 ppm SO, have been identified (Eschenbruch, 1974).
Sulfite serves several functions in wine, including antimicrobial functions,
prevention of browning, and binding of acetaldehyde (Eschenbruch, 1974).
However, sulfite can be detected organoleptically if the concentration becomes
too high; the threshold is thought to be about 50 ppm as free sulfite. Because of
its adverse effects on the organoleptic quality of the wine and the potential for
abuse of sulfites in making wine from inferior grapes, many countries have
imposed strict limitations on the amount of residual SO, allowed in wine. In the
Federal Republic of Germany, for example, the limits are 50 ppm of free sulfite
and 300 ppm for total sulfite in wines of the Qualitatswein class. In the United
States, the upper limit is 350 ppm as total residual SO,, although BATF is
proposing an upper limit of from 125 to 175 pprn (Anonymous, 1984).
Consequently, the formation of sulfite by yeasts must be critically controlled.
The choice of yeast strains is important since they can vary by an order of
magnitude in their capacities for sulfite formation (Dott et al., 1976; Eschen-
bruch, 1974; Rankine and Pocock, 1969). The high sulfite formation by certain
yeast strains can be attributed to several metabolic differences related to sulfite
SULFITES IN FOODS 7
metabolism (Dott et al., 1977; Eschenbruch and Bonish, 1976; Heinzel and
Truper, 1976). However, both low- and high-sulfite-forming strains are equiv-
alent in their abilities to reduce sulfite to the obnoxious sulfides (Dott and
Truper, 1976). The extent of sulfite formation is also related to other factors,
including the amount of sulfite-bindingcompounds produced in the fermentation
(Rankine and Pocock, 1969; Weeks, 1969). Acetaldehyde, pyruvic acid, and a-
ketoglutaric acid bind SO, and serve to control the quantities of free SO, in the
fermentation medium (Burroughs and Sparks, 1973a-c; Rankine and Pocock,
1969; Weeks, 1969). Only the free SO, has antimicrobial properties.
The situation is much the same in beer except that lower levels of SO, are
produced during beer fermentation (Hysert and Morrison, 1976). The SO, is
derived mainly from sulfate and also serves as a precursor for sulfides.
Wine and beer cannot be made without formation of sulfites. In beer, residual
total SO, levels ranged from 0.2 to 11 ppm in the absence of added SO, in one
study (Hysert and Morrison, 1976), although higher natural levels of formation
might be expected to occur. Much of the residual sulfite in beer is in the
combined state (Chapon et al., 1982). In wine, natural SO, formation can
account for 15-125 ppm of residual SO, in the finished product. In other food
products fermented by yeasts, we would expect that SO, formation from sulfate
would occur naturally, although we are not aware of studies confirming this
suspicion.
C. HISTORY OF USE OF SULFITING AGENTS

AS FOOD INGREDIENTS
The sulfiting agents have enjoyed a long history of effective use as food and
drug ingredients. Ancient Greeks wrote about the use of SO, for the fumigation
of houses. The Romans and Egyptians are supposed to have used SO, for the
sanitation of wine vessels (Roberts and McWeeny, 1972). Its use as a food
preservative dates to at least 1664 when cider was added to flasks while they still
contained SO, (Evelyn, 1664). The inorganic sulfites appeared as food additives
at a much later date. The first years of use of the various inorganic sulfiting
agents in the United States are as follows: sodium bisulfite, 1921; sodium sulfite,
1930; potassium and sodium metabisulfite, 1939 (Subcommittee on Review of
the GRAS List, 1972). The sulfiting agents were first used in wine and beer.
Among nonalcoholic products, the sulfiting agents were first used on dried fruits
and vegetables in all likelihood. However, their use in foods spread rapidly as a
consequence of the absence of toxic hazards and their widespread functional
effectiveness. In the decade between 1960 and 1970,.a 30-70% increase in the
amounts of several sulfiting agents used annually in the United States was ob-
served (Subcommittee on Review of the GRAS List, 1972), a testament to the
TABLE I11
FOOD USE OF SULFlTING AGENTS
(UNITED STATES, 1976)
Amount produced
Sulfiting agent (Ib)
Sodium bisulfite 4,900,000

Sodium metabisulfite 92,000
Potassium metabisulfite 220,000
Sulfur dioxide 2,200,000
Sodium sulfite 15,000
growing utilization of these additives. Table 111 contains data on the production
of sulfur dioxide and the inorganic sulfiting agents for use in foods. Foods
represent one of the larger uses for these chemicals. SO, and sodium bisulfite are
produced and used in far larger quantities than most of the other sulfiting agents.
D. CURRENT FOOD APPLICATIONS
1. Overview of Current Applications
Sulfiting agents are used in foods for many important purposes: the inhibition
of nonenzymatic browning, the inhibition of various enzymatic reactions includ-
ing enzymatic browning, inhibition and control of microorganisms, an antioxi-
dant and reducing agent including dough conditioning, a bleaching agent, a
processing aid, and several secondary uses including pH control agent and sta-
bilizing agent. Each of the general categories will be discussed with a brief
explanation of the mechanism of action of the sulfiting agents in effecting these
changes. As might be expected for any group of substances that possesses so
many useful properties, an enormous number of specific applications have been
found for sulfiting agents in foods. Later in this section, we will make an attempt
to identify these applications and the treatment levels associated with each ap-
plication. Other uses for the sulfiting agents have been devised, and these uses
will be described, although we are not certain that they are being used in the food
industry. Finally, the sulfiting agents provide other benefits in foods beyond
those which are readily identified with the sulfiting agents. These benefits will be
identified and discussed.
Several previous reviews on the applications for sulfiting agents in foods have
appeared (Green, 1976; Joslyn and Braverman, 1954; Roberts and McWeeny,
1972; Schroeter, 1966). Based on our current knowledge, these reviews are
inadequate and should be considered to be out of date (Joslyn and Braverman,
SULFITES IN FOODS 9
1954; Schroeter, 1966), oriented toward the British food industry (Green, 1976;
Roberts and McWeeny, 1972), or incomplete. Many additional applications of
the sulfiting agents have now been identified, although the previous reviews do
provide much valuable information on some of the major uses of the sulfiting
agents in foods.
2 . Inhibition of Nonenzymatic Browning
Nonenzymatic browning is a term used to describe a family of diverse reac-

tions that commonly involve the formation of carbonyl intermediates and brown,
polymeric pigments. Examples include the reactions between amino acids and
reducing sugars and carmelization of sugars. The chemistry of the reactions
involved is complex and not completely understood. An excellent review of the
chemistry of nonenzymatic browning and the effects of the sulfites on these
reactions was prepared by McWeeny et al. (1974). The sulfites can be used to
control nonenzymatic browning because of their ability to react with the carbonyl
intermediates. A variety of carbonyl intermediates can be formed during the
nonenzymatic browning process, including reducing sugars, simple carbonyls,
dicarbonyls, and a,P-unsaturated carbonyls. The sulfites can react with all of
these intermediates and thus block formation of the brown pigments. Wedzicha
et al. (1984) developed a kinetic model for the inhibition of nonenzymatic
browning by sulfites. Reaction of sulfites with the carbonyls generated by non-
enzymatic browning accounts for most of the loss of sulfites in dehydrated
vegetables (Wedzicha et al., 1984). Some of the sulfite-carbonyl reaction prod-
ucts are more stable than others. The sugar hydroxysulfonates formed between
reducing sugars and sulfites are the least stable, although they are quite stable at
acid pHs. The sulfonated carbonyls formed on reaction of the sulfites with the
a,@-unsaturatedcarbonyls are extremely stable and the reaction is generally
considered to be irreversible. The differences in stability of the sulfite addition
products can influence the effectiveness of the sulfites in certain food applica-
tions. For example, sulfite can almost totally inhibit nonenzymatic browning of
glucose-glycine solutions because of the irreversible reaction of the sulfites with
the a$-unsaturated carbonyl intermediates of this reaction (Wedzicha and Mc-
Weeny, 1974a). The glucose-glycine system typifies the situation that occurs in
dehydrated potatoes. A kinetic model for the glucose-glycine reaction and its
inhibition by sulfites has been developed by Wedzicha (1984). On the other
hand, sulfites can only retard the formation of browning pigments in the ascorbic
acid-glycine reaction because the principal intermediates of this reaction are
dicarbonyl compounds which react reversibly with the sulfites (Wedzicha and
McWeeny, 1974a). The ascorbic acid-glycine system is typical of the situation
existing in fruit juices and drinks.
Sulfites find wide use as inhibitors of nonenzymatic browning. They have
been used for this purpose to control discoloration of wines, dried fruits, dehy-
drated vegetables, dehydrated potatoes, coconut, pectin, some varieties of vin-
egar, and white grape juice. Sulfites are also used to control the commercial
carmelization process. In warmer climates, sulfites can be used to control non-
enzymatic browning in fruit juices and drinks (Joslyn and Braverman, 1954).
Sulfites are also used to control juice color formation in the production of beet
sugar (McGinnis, 1982).
3. Inhibition of Various Enzymatic Reactions
SO, and sulfites can act as inhibitors of numerous enzymatic reactions, includ-
ing polyphenoloxidase, ascorbate oxidase, lipoxygenase, peroxidase, and thia-
mine- dependent enzymes. The actions of the sulfiting agents on oxidizing
enzyme systems have been reviewed by Haisman ( 1974).
Inhibition of polyphenoloxidase is useful in the control of enzymatic brown-
ing. Polyphenoloxidase catalyzes the oxidation of mono- and ortho-diphenols to
quinones. The quinones can cyclize, undergo further oxidation, and condense to
form brown pigments. The mechanism of action of the sulfites in preventing
enzymatic browning is not known, but very likely involves several different
types of actions. Sulfites may directly inhibit the enzyme; potassium meta-
bisulfite has recently been shown to inhibit strawberry polyphenoloxidase at 10
mM concentrations (Wesche-Ebeling and Montgomery, 1983). Sulfites may also
interact with the intermediates in the enzymatic browning reaction and prevent
their participation in the reactions leading to formation of the brown pigments.
For example, sulfites may combine with the quinones and prevent their participa-
tion in the further oxidation, cyclization, and condensation reactions. Evidence
for the formation of quinone-sulfite complexes has been reviewed (Haisman,
1974). Alternatively, the sulfites may simply act as reducing agents promoting
the reaction of the quinones back to the original phenols. The level of sulfites
necessary to prevent enzymatic browning depends on the nature of the available
substrate. When only monophenols such as tyrosine are present, fairly low levels
of sulfite are effective. Potatoes are an example of this situation. When diphenols
are present, much higher concentrations of sulfites are necessary. An example of
this situation would be guacamole. The sulfites do not irreversibly inhibit the
enzymatic browning reaction so the required concentrations are also dependent
on the length of time that the reaction must be inhibited.
Inhibition of enzymatic browning is the primary reason for using sulfites in
salad bar items, including cut fruits, lettuce, and guacamole. Sulfites have also
been used to prevent enzymatic browning in prepeeled potatoes, sliced potatoes,
cut apples and other fruits supplied to the baking industry (Ponting et al., 1971),
fresh mushrooms (Komanowsky et al., 1970), and table grapes (Nelson, 1983).
SULFITES IN FOODS 11
A similar reaction occurs in shrimp where enzymatic tyrosine oxidation leads

to black spot formation. The reaction is catalyzed by tyrosinase, a type of
polyphenoloxidase. Black spot formation in shrimp can be controlled by the
addition of sulfites (Fieger, 1951).
Sulfites can also prevent the oxidation of ascorbate by ascorbate oxidase and
other enzymes. Ascorbate levels decrease very quickly following the maceration
of plant tissues due to the action of ascorbate oxidase. Sulfite addition preserves
ascorbate and can be used in potato, pumpkin, cauliflower, tomato, and green
and red pepper products (Haisman, 1974).
Sulfites can also inhibit lipoxygenase, an enzyme known to cause formation of
off-flavors during postharvest storage of vegetables such as peas (Haisman,
1974). Treatment with sulfites will prevent formation of these off-flavors, an
added benefit to their use in dehydrated peas and other vegetables.
Anaerobic bacterial fermentation can be inhibited in grape juice by sulfites.
This inhibition is essential to the production of wines. The mechanism of this
inhibition is not entirely understood, but it is partially due to the destruction of
thiamine, which serves as an essential cofactor for several of the fermentative
enzymes (Haisman, 1974). These enzymes are thus inhibited.
4. Inhibition and Control of Microorganisms
The sulfites play crucial roles in the inhibition of bacteria in several food pro-
cesses. In winemaking, the sulfites are employed to prevent undesirable bacterial
fermentation of the grape or fruit juice. Sulfites are also essential in the corn
steeping process used to facilitate removal of the corn starch; the sulfites prevent
bacterial growth in the steep liquor (Schroeter, 1966). The application of sulfites
to table grapes is critical to prevent bacterial and mold growth (Nelson, 1983;
Nelson and Ahmedullah, 1973, 1976). Although not a common practice in the
United States, sulfites have been widely used to prevent mold damage in fruits
prior to jam production (Roberts and McWeeny, 1972). Sulfites have also found
use in the prevention of postharvest deterioration of fruits used for the production
of juices (Moms et a f . , 1979).
The use of sulfites as antimicrobial agents has been reviewed by Roberts and
McWeeny (1972), Joslyn and Braverman (1954), and Ingram (1959). The sul-
fites are selective antimicrobial agents with more inhibitory effect on acetic acid
bacteria, lactic acid bacteria, and various molds than on yeasts (Joslyn and
Braverman, 1954). This selectivity enhances their value in the control of undesir-
able fermentation in winemaking. The mechanism of the antimicrobial action of
the sulfites is not well understood. However, several factors are known to control
the antimicrobial efficacy of the sulfites. One of the more important factors is pH
which controls the form of sulfite present in the food. Apparently, H,SO, is the
active form of the sulfites in terms of their antimicrobial actions (Carr et al.,
1976; Ingram, 1959), so lower pHs enhance the antimicrobial effect. The com-
bination of sulfites with food components also affects their antimicrobial activity
(Ingram, 1959; Joslyn and Braverman, 1954). The sulfite adducts have no anti-
microbial activity. Consequently, more sulfite is required to preserve a glucose
syrup than a sucrose syrup, since sulfites will combine with glucose but not
sucrose (Ingram, 1959). Considerable sulfite must be added to wine because of
the binding of the sulfites to fermentation products such as acetaldehyde. The
volatilization of SO, from acidic products also affects the level retained for
antimicrobial action.
Sulfites can have some detrimental effects as a result of their antimicrobial
actions. In red wines, high levels of SO, inhibit the desirable malolactic fermen-
tation, which serves to reduce the acidity of wines produced in cool regions (Liu
and Gallander, 1983).
Although we know of no practical use of this antimicrobial activity, sulfites
also inactivate certain types of enteroviruses including poliovirus type I, cox-
sackievirus type A9, and echovirus type 7 (Salo and Cliver, 1978).
5. Antioxidant and Reducing Agent Uses
The antioxidative effects of the sulfites are partially responsible for their
preserving effect on ascorbate and their inhibition of nonenzymatic and en-
zymatic browning. The ability of the sulfiting agents to promote the reduction of
the oxidized quinones to reduced phenols is one of the mechanisms available for
the inhibition of these processes by the sulfites. Sulfites also prevent the oxida-
tion of essential oils and carotenoids, which would generate off-flavors (Baloch
et al., 1977; Roberts and McWeeny, 1972). A major function of SO, in beer is
the inhibition of oxidative changes that are considered undesirable to flavor
development (Roberts and McWeeny, 1972; Schroeter, 1966).
Sulfites are widely-used as dough conditioners in the baking industry for
biscuits, crackers, cookies, and frozen pizza doughs and pie crusts. In these
products, sulfites act by breaking the disulfide bonds in the gluten fraction of the
dough (Wade, 1972). The sulfites also promote disintegration of the protein
matrix during the corn steeping process, which facilitates rapid hydration, soft-
ening of the kernel, and extraction of the starch (Schroeter, 1966). The sulfites
may exert this action via their ability to reduce disulfide bonds, although we
know of no direct proof for this possibility. SO, has also been used to improve
the extraction of pectins from various sources through its ability to depolymerize
the pectins (Roberts and McWeeny, 1972).
6. Bleaching Agent Uses
The major application of the bleaching properties of the sulfites is the bleach-
ing of cherries for the production of maraschino cherries and g l a d fruit products
(Josyln and Braverman, 1954; Weigand, 1946). The sulfiting agents are also
reported to bleach pectins (Roberts and McWeeny, 1972). The uniformity and
translucency of color of orange, lemon, grapefruit, and citron peel are improved
by storage in a sulfite brine (Cruess and Glickson, 1932). Sulfur dioxide can also
be used as a bleaching agent for food starches (Table I). The bleaching of table
grapes during sulfite fumigation is considered detrimental to quality (Nelson,
1983).
7. Use as a Processing Aid
Many of the applications of sulfites fall into the category of processing aids.
This particular category of use for sulfiting agents is difficult to define and
variations probably exist in its definition within the food industry. Obviously,
sulfite residues can originate from the use of sulfited products in the formulation
of the end product. Examples would include the use of beet sugar or corn syrup
in a variety of products and the use of maraschino cherries in fruit cocktail.
Typically, SO, residue levels from such uses would be rather low. Further
investigation of the use of sulfiting agents as processing aids will be necessary to
obtain a better picture of the extent of such uses and their contribution to con-
sumer exposure.
8. Secondary Uses
This category of uses of the sulfiting agents is diverse because these additives
have many desirable secondary benefits beyond the primary reasons for their use.
Examples would include their facilitation of corn starch extraction (Schroeter,
1966), a secondary benefit to the primary purpose of preventing microbial
growth in the corn steep liquor. Another example would be the control of excess
alkalinity and the improvement in boiling properties of beet sugar juice, a sec-
ondary benefit to the primary purpose of control of color formation in the juice
(McGinnis, 1982). Many additional examples could be selected.
9. Specific Applications and Treatment Levels
The above discussion clearly shows that sulfiting agents are used in the food
industry for a variety of products and for many different reasons. The Federation
of American Societies for Experimental Biology (FASEB) panel attempted to

identify these applications and the residue levels resulting from each use (Life
Sciences Research Office, 1985). This information may not be representative of
the entire food industry, since variations exist in the use of sulfiting agents, the
type of sulfiting agents employed, the treatment levels, and the means of apply-
ing the sulfiting agents to the foods, all of which would affect residual levels.
Sulfite uses have been identified in baked goods and baking mixes, alcoholic and
nonalcoholic beverages, coffee and tea, condiments and relishes, dairy product
analogs, prepared fish and shellfish products, fresh fish and shellfish, fresh fruits
and fruit juices, fresh vegetables, gelatins, grain products, gravies and sauces,
jams and jellies, nuts and nut products, processed fruits and fruit juices, pro-
cessed vegetables and vegetable juices, snack foods, soups and soup mixes,
sugar, and sweet sauces, toppings, and syrups. These categories correspond to
those listed in the CFR (21CFR 170.3). In Table IV, specific uses of the sulfiting
agents within each of these categories are identified, and reported residual levels
and exposure estimates are given (Life Sciences Research Office, 1985); most of
the information in Table IV was obtained from the FASEB compilations (Life
Sciences Research Office, 1985). Some of the information in Table IV needs to
be verified for accuracy, but it is probably the best and most complete survey of
sulfite use ever conducted. Also, the uses and residual levels may not be repre-
sentative of the entire industry. A major deficiency has been the lack of analyses
of sulfited foods at the point of consumption. Storage, processing, and prepara-
tion can affect residual sulfite levels in the product prepared for consumption.
Further research will be needed to determine, with greater accuracy, actual
consumer exposure to sulfites.
10. Other Uses for Sulfiting Agents in Foods
Certain other uses have been developed for the sulfiting agents in foods. We
are not certain that these processes are actually being used in the food industry,
but they are feasible. Two examples will be cited, although many more appear in
the literature. A procedure for improved color retention in canned garbanzo
beans that involves a presoak in NaHSO, has been developed (Daoud et al.,
1977; Luh et al., 1978). The pink discoloration noted with certain varieties of
canned pears can be prevented by use of SO, (Chandler and Clegg, 1970).
11. Additional Benefits of Sulfiting Agents in Foods
Sulfites provide additional benefits in foods beyond those already discussed.

To our knowledge, they are not used in foods for these purposes, so these
benefits might best be classified as fortuitous or potential uses. The carcinogenic
TABLE IV
ESTIMATED INTAKE OF SULFITES FROM VARIOUS FOODS"
Estimated level
in product
as consumedb Food intake Sulfite intakec
Category Subcategory (ppm SO2) (g/capita/day) (mg/capita/day)
Baked goods Cookies 5 6.2 0.031

and baking mixes Cake with dried carrots 10 0.1 0.001
Crackers 5 2.9 0.014
Sheeting doughs <1.5 - -
Pie dough <6 - -
Pizza ciust 5 1.3 0.007
Pie crust 5 I .7 0.009
Tortilla shells 5 1.6 0.008
Beverages, Total 3.3 139 0.46
nonalcoholic Cola and pepper 4
Lemon-lime 1
Orange 1
Root beer 4
Ginger ale 1
Grape 1
Juice-containing carbonated 4
beverage
Beverages, Beer 10 38 0.38
alcoholic Wine 150 5.4-24.5 0.81-3.68
Coffee and tea Instant tea 5-6 24 0.14
Liquid concentrated tea 5 1.2 0.006
Tea leaves - 95 -
Condiments Olives 2 0.1 0.002

Pickles/relishes 30 0.25 0.008
Salad dressing mix (dry) 10 0.08 0.001
Vinegar
Malt vinegar 10 0.06 0.001
Wine vinegar 15 0.24 0.018
Dairy analogs Filled milk 200 0.15 0.03
Fish and shellfish Dried cod 10 12 0.12
Shrimp 4-36 0.6 -
Fresh fruit Fruit salad 5 42.6 0.213
Grapes 1-5 3.9 -
Dried fruitd Total 1200 0.49 0.59
Fruit juices Apple concentrate, imported 5 42.6 0.243
Cheny-beny 10 0.2 0.002
Grape, red or purple 3 3 0.009
Grape, white, white sparkling, 85 0.3 0.025
pink sparkling, or red spark-
ling
(continued)
TABLE 1V (Conrinued)
Estimated level
in product
Category Subcategory (ppm SO2) (gkapitalday) (mg/capita/day)
Lemon, nonfrozen 800 0.12 0.096

Lime, nonfrozen 160 0.04 0.016
Frozen fruit Unspecified 5 0.5 0.025
Canned fruit Maraschino cherries 50 0.3 0.015
Gelatin Total 5 6.6 0.033
Flavored mix 4 - -
Unflavored mix 5 - -
Grain products Corn starch and modified corn 20 3.7 0.074

starch
Hominy 20 0.1 0.002
Spinach pasta 5 0.1 0.001
Gravies, sauces Meat, tomato, milk, buttery, 75 3 0.225
and specialty
Jams and jellies Domestic 5 5.7 0.029
Imported 13.6 0.06 0.001
Pectin > 10-50 - -
Nut products Coconut 5 0.5 0.003
Protein isolates, soy 2-5 0.25 0.017
Snack foods Apple bits - - -
Crackers, filled 0.5 0.29 0.0001
Corn-based snacks 0.5 0.88 0.0005
Pretzels - - -
Soft candy Caramel - - -
soups Dry mix 0.5 9.3 0.005
Sugars and frostings Sugars, average 7 99 0.70
Sweet sauces Corn syrup 30 26 0.78
and syrups High fructose corn syrup 3 50 0.150
Fruit topping 60 3.4 0.203
Maple syrup 20 3 0.06
Molasses 125 0.27 0.033
Fresh vegetables Total 16 39.1 0.63
Mushrooms 13 0.2 -
Salad bar lettuce 950 - -
Cole slaw < 10-250 - -
Guacamole - - -
Potatoes, whole, peeled - - -
Canned vegetables Total 5 65 0.325
Potatoes 10 0.27 -
Pickled cocktail onions 30 - -
Pickled peppers 30 - -
Sauerkraut (in glass) 30 0.12 -
Sauerkraut (in cans) 12 1.1 -
TABLE IV (Continued)
Estimated level
in product
Category Subcategory (ppm SO*) (glcapitalday) (mg/capita/day)
Dried vegetables Potatoes I5 3.2 0.24

Frozen vegetables Potatoes 20 1.7 0.034
Vegetable juices Total 5 10 0.05
Sauerkraut juice 100 - -
a Compiled largely from the report entitled “The Reexamination of the GRAS Status of Sulfiting Agents,”
prepared by the Life Sciences Research Office (Federation of American Societies for Experimental Biology),
January, 1985.
Reported as ppm of SO, equivalents using total SO2 data in most cases.
Reported as SO2 equivalents.
Includes dried apples, apricots, peaches, golden raisins, cherries, figs, pears, tropical fruit (pineapple,
mango, papaya), dried fruit mixes, cake with dried fruit, and dried fruit fillings. Exceptions are dark raisins and
prunes which are not sulfited.
mycotoxins, aflatoxins B, and G,, can be degraded by bisulfite, although rather

high levels are required (Doyle and Marth, 1978a,b). The complete destruction
of aflatoxin B, in corn requires up to 10% bisulfite treatment for 72 hr, but
chickens fed this corn were unaffected (Hagler et al., 1982). The bisulfite addi-
,
tion product with aflatoxin B has been identified (Hagler et al., 1983). Naturally
occurring aflatoxin M, in milk can be destroyed by sulfite addition also (Ap-
plebaum and Marth, 1982). Burroughs (1977) showed that the mycotoxin, pa-
tulin, could be destroyed by SO,, but concentrations of 2000 ppm were neces-
sary to remove 15 ppm patulin from apple juice and cider. In brewing, the
presence of SO, in production malts seems to control the formation of N-nitro-
sodimethylamine (NDMA) in the malt (Lukes et al., 1980). Malt systems con-
taining 30 mg/kg residual SO, always had less than 10 pg/kg NDMA. At lower
SO, levels, progressively higher concentrations of NDMA were found. The
mutagenicity of coffee can be suppressed by sulfites in the concentration range of
about 200 ppm (Suwa er al., 1982).
E. METHODS FOR MEASUREMENT OF SULFITE RESIDUE LEVELS
1 . Free versus Total SO,
Numerous methods for SO, and sulfite residue analysis have been developed
over the years, and it is beyond the scope of this review to provide a critical
analysis of all of these methods. However, comments must be made on the
relative merits of measuring total SO, versus free SO,.
Sulfites exist in foods as sulfurous acid, inorganic sulfites, and a variety of

forms of combined sulfites. Complex equilibria dependent on a number of fac-
tors control the amount of sulfite in each of these states, as will be discussed in
the next section. Methods for the measurement of free SO, are aimed at detection
of undissociated sulfurous acid, bisulfite ions, and sulfite ions. Methods for the
measurement of total SO, are aimed at detection of these substances plus some of
the combined forms of the sulfites. Generally, the methods for analysis of total
SO, can be subdivided into two groups: those in which the combined SO, is
liberated by distillation from acid, and those in which the combined SO, is
liberated by treatment of an extract with excess alkali. SO, will not be liberated
from all forms of combined sulfite by either of these treatments; some combined
sulfites are quite stable. The levels of combined sulfites are not included in some
methods of analysis as a distinct determination. They are often calculated from
the differences between total SO, and free SO,, and thus are underestimates
representing only the dissociable forms of combined sulfites. Many methods
exist for the measurement of free, total, and combined SO,. The most widely
used methods were developed many years ago and were critically reviewed by
Joslyn and Braverman ( 1954).
2 . Analysis of Free SO,
Methods for the measurement of free SO, are largely variations on the original
iodometric titration method developed by Ripper (1892). The titration is done
following acidification which is relied upon to reduce the rate of dissociation of
combined sulfites. Several problems can occur with the measurement of free SO,
by these procedures: Iodine-reducing substances other than SO, may be present,
recombination of SO, with carbonyls may occur, oxidation to sulfate may occur,
some weakly bound forms of SO, may be dissociated, such as the anthocyanin
complexes in wine, and colored substances may interfere (Bruno et al., 1979;
Joslyn and Braverman, 1954; Ponting and Johnson, 1945; Vahl and Converse,
1980). Oxidation to sulfate (Joslyn and Braverman, 1954) and the presence of
other iodine-reducing substances (Ponting and Johnson, 1945)can be controlled.
However, the method is also subject to considerable error and poor precision, at
least for the analysis of SO, in wine (Vahl and Converse, 1980). We have found
this method to be unsuitable for the determination of free SO, in many foods.
Fujita et al. (1979) noted that extreme care must be taken to prevent dissociation
of combined sulfites in these methods. Improved methods, including aeration-
oxidation procedures (Buechsenstein and Ough, 1978; Fujita et al., 1979;
Ogawa et al., 1979; Mitsuhashi et al., 1979; Rankine and Pocock, 1970), polar-
ographic methods (Bruno et al., 1979), flame photometric detection by gas
chromatography (Hamano et al., 1979; Mitsuhashi ef al., 1979), ion chro-
matography (Anderson et al., 1983), and enzymatic assays with sulfite oxidase
(Beutler, 1984), have been developed, but not widely tested with a variety of
foods.
3. Analysis of Total SO,
The most commonly used method for analysis of total SO, residues is the
Monier-Williams method (Monier-Williams, 1927), an acidic distillation pro-
cedure. The Monier-Williams method for many years has been considered to be
the most reliable method for SO, analysis in foods (Joslyn and Braverman,
1954). However, the method is extremely time-consuming and laborious. That
has led to considerable effort in the search for suitable alternatives. Although the
method is reproducible and apparently free from most types of interference, it is
not the perfect method. First, it does not truly measure total SO,, since some
forms of combined sulfites are not dissociated during the acidic distillation
procedure (Jennings et al., 1978; Wedzicha and Bindra, 1980). Also, the
Monier-Williams procedure can be subject to interference by other sulfur com-
pounds in foods (Mitsuhashi et al., 1979; Wedzicha and Bindra, 1980). Several
improved methods for the analysis of total SO, have been developed, including
alkali treatment coupled with idometric titration (Ponting and Johnson, 1945),
the Rankine aeration-oxidation method (Buechsenstein and Ough, 1978; Fujita
et al., 1979; Mitsuhashi et al., 1979; Ogawa et al., 1979; Rankine and Pocock,
1970), polarographic methods (Bruno et al., 1979), colorimetric procedures
(Jennings et al., 1978; Nury et al., 1959), combustion in oxygen with measure-
ment of the sulfur in the gaseous products and the residue (Wedzicha et al.,
1984), sulfite oxidase assays (Beutler, 1984), and flame photometric detection
after gas chromatography (Hamano et al., 1979; Mitsuhashi et al., 1979). These
methods have generally been shown to yield results similar to the Monier-
Williams method. Several other methods, including a microdiffusion method
(Adachi et al., 1979), a direct colorimetric method (Adachi et al., 1979), and
gas-sensing probes (Jennings et al., 1978), have been found to have inaccuracies
or limited uses. Some of these alternative methods allow simultaneous determin-
ation of free, combined, and total SO, (Bruno et al., 1979; Buechsenstein and
Ough, 1978; Fujita et al., 1979; Mitsuhashi et al., 1979; Ogawa et al., 1979;
Ponting and Johnson, 1945; Rankine and Pocock, 1970). However, none of them
is suitable for the measurement of all forms of combined sulfite.
4. A Critical Appraisal of Sulfite Analysis
For unknown reasons that probably date back many years, the measurements
of free and total sulfite residues in foods are referred to as free and total SO,
analyses. The reason probably relates to the release of SO, under the conditions
of the assay. However, SO, is not the form that actually exists in foods. The free
SO, methods are actually detecting residues of free inorganic sulfite salts. It
would be preferable to refer to them as assays of free sulfite (or free sulfite as
SO,) rather than free SO,. The total SO, methods are detecting the free sulfite
residues as well as some of the combined forms of sulfite. The combustion
method described by Wedzicha et al. (1984) may detect most of the combined
forms of sulfite. These methods should be referred to as assays of total sulfite (or
total sulfite as SO,) rather than total SO,, although the use of the adjective total
may be misleading, since not all forms of combined sulfites can be detected with
these assays.
Considerable data exist in the literature on residual SO, levels in foods (some
of that data can be found in Table IV). However, for several reasons, we have
some reservations about these data. Many methods exist for the measurement of
residual sulfite levels in foods, and correlations between the various methods
have not been established for most foods. Therefore, it is difficult to evaluate the
validity of some of the published residue data. Some of the methods used to
generate these residue data have subsequently been shown to give erroneous
results. Second, very little residue data are available for sulfited foods obtained
from the marketplace. Much of the available data were obtained from products
sampled immediately after processing. Therefore, the effects of storage and any
differences with standard, present-day commercial practices have not been taken
into account. Much of the available residue data are from fairly old studies, and
treatment conditions have probably changed in the intervening years. As men-
tioned previously, the effects of preparation on residual sulfite levels have essen-
tially been ignored in previous work.
However, the most important issue surrounds the question of what should
actually be measured. Some of the previous data are for free SO, and some are
for total SO,. As detailed in Section 111, we speculate that the free inorganic
sulfites are much more likely to be implicated in the toxic and asthmatic reactions
than are the organic sulfite adducts. For consumer exposure assessments, one
would ideally wish to measure exposure to the forms of sulfite that cause toxic or
hypersensitivity reactions, most likely the free inorganic sulfite residues or the
free sulfite residues plus those combined forms of sulfite that would be expected
to release SO, in vivo during the digestive or metabolic processes (see Section
111). Most of the exposure assessments have used total SO, residues. Yet, the
conditions of the Monier-Williams distillation procedure are much more severe
than any conditions that would be encountered in vivo. Therefore, the Monier-
Williams procedure may detect some combined forms of sulfite that would not be
expected to release SO, in vivo. More information is needed on the in vivo
stability of various combined forms of sulfite in comparison to their recovery in
the Monier-Williams or other procedures for total sulfite analysis. While the
Monier-Williams procedure may detect excessive amounts of SO, for accurate
exposure assessments, the free sulfite analyses may not be suitable because they
do not detect the combined forms of sulfite that release SO, in vivo.
The confusion is magnified by the numerous variations and modifications of
the procedures for the determination of free and total sulfite. The alternative
and/or modified procedures in many cases yield different results for the same
sample. Standardized Association of Official Analytical Chemists (AOAC) pro-
cedures exist for both free and total SO, analysis (Horwitz, 1975), but many
analysts use modifications of these methods. Therefore, it is difficult to compare
the data of one analyst with those of another. This uncertainty further confuses
the situation for those performing exposure assessments.
F. CHEMISTRY OF SULFITES AND FATE IN FOODS
I . EfSect of pH on Su&tes
Sulfur dioxide dissolves readily in water-producing sulfurous acid, H,SO,.

On treatment with alkali, sulfurous acid yields sulfites, bisulfites, and meta-
bisulfites. These inorganic forms of sulfites are in equilibrium with one another
in aqueous solutions and the concentration of each species is dependent on pH.
At high pHs, SO$- is the predominant species, while at very low pHs, H,SO,
predominates. At intermediate pHs, HSO, predominates, reaching a maximum
concentration at pH 4.0. SO, can be evolved from H,SO,, but only at acid pHs.
Table V shows the maximum percentage of SO, that could be liberated at various
pHs. Note that no SO, can be evolved from a solution until the pH drops to pH
4.0 or below. A more complete discussion of the distribution of the various
forms of sulfite as a function of pH can be found elsewhere (Green, 1976; Joslyn
and Braverman, 1954).
TABLE V
PERCENTAGE OF SO, AT VARIOUS pHs"
PH Free SO2 (%)
I 86
2 37
2.5 16
3 6
4 0.5
From Green (1976).

2. Fate of Sulfites in Foods
The sulfites react readily with a variety of food constituents, including al-
dehydes, ketones, reducing sugars, unsaturated organic compounds, browning
intermediates, proteins, and anthocyanins, to name but a few. The extent of
reaction is dependent on the pH, temperature, concentration of sulfite, and the
reactive components of the food matrix. An equilibrium always exists between
the combined and free forms of the sulfites, although some of the reactions are
virtually irreversible, while others are more readily reversible (Wedzicha et al.,
1984). The reactions remove free sulfites from the food which often diminishes
their effectiveness in the food product. The dissociable, combined forms of
sulfite can serve as a reservoir for free sulfite, but the irreversible reactions
remove sulfite permanently from the pool of free SO,. As we noted earlier, most
of the desirable actions of the sulfites are dependent on the free forms; the
combined sulfites are usually ineffective. Therefore, treatment levels for specific
foods have historically been chosen to provide an active, residual level of free
SO, throughout the typical shelf life of the product.
3 . Reactions of Sulfites with Carbonyls
Sulfites are known to have a particular affinity for reactions with aldehydes
and ketones. The primary products of these reactions are hydroxysulfonates
(Joslyn and Braverman, 1954). Burroughs and Sparks (1973a-c) have made a
very thorough study of the reactions between carbonyls and sulfites. The reaction
rates between carbonyls and sulfites are fast, and the equilibrium constants
overwhelmingly favor the hydroxysulfonates in the range of pH 1-8 (Burroughs
and Sparks, 1973a). At higher pHs, more dissociation occurs (Burroughs and
Sparks, 1973a). Temperature changes in the range of 0-60°C have little effect on
the stability of acetaldehyde hydroxysulfonate, but pyruvic acid hydroxysulfo-
nate shows progressively greater dissociation at higher temperatures (Adachi et
al., 1979). Burroughs and Sparks (1973b,c) have succeeded in developing a
model for the sulfite-binding properties of wines and ciders on the basis of the
concentrations of various types of carbonyl compounds. Acetaldehyde is the
primary sulfite-binding substance in these beverages, since it is a primary prod-
uct of the fermentation process (Burroughs and Sparks, 1973b). The ability of
sulfites to inhibit the nonenzymatic and enzymatic browning reactions is largely
due to their reactions with the carbonyl intermediates produced in these reactions
(Haisman, 1974; McWeeny et al., 1974). Wedzicha et al. (1984) demonstrated
that the reaction of sulfites with carbonyls generated from nonenzymatic brown-
ing was the major reaction of sulfites in dehydrated vegetables.
The hydroxysulfonates of carbonyl compounds are rather stable reaction prod-
ucts. The most stable carbonyl hydroxysulfonates would be those formed with
the a,P-unsaturated carbonyl intermediates of the browning reaction (McWeeny

et al., 1974). However, acetaldehyde hydroxysulfonate should be considered an
extremely stable reaction product (Adachi et al., 1979; Burroughs and Sparks,
1973a). Although some of the other carbonyl hydroxysulfonates are somewhat
less stable, such as the pyruvic acid product (Adachi et a l . , 1979; Burroughs and
Sparks, 1973a), this entire class of reaction products is stable. They would not be
expected to dissociate to any great extent in the stomach, and dissociation in the
small intestine would not even be particularly rapid.
4 . Reactions of Sulfites with Reducing Sugars
The sulfites are also capable of reactions with reducing sugars, such as
glucose. Sugar hydroxysulfonates are not formed as readily as carbonyl hy-
droxysulfonates; a considerable molar excess of reducing sugar is usually re-
quired (Adachi et al., 1979). The glucose hydroxysulfonates are also much less
stable than their carbonyl counterparts (Green, 1976; Joslyn and Braverman,
1954). At the neutral pH of 7.0, the reaction between sulfites and glucose would
be very rapid, but dissociation would predominate, leaving the bulk of the sulfite
in the free form (Green, 1976). At pH 4 to 5 , the reaction rate would still be
fairly fast and dissociation would be much slower, thus favoring the combined
form (Green, 1976). At pH 2, the reaction rate would be slow but the product,
once formed, would be stable (Green, 1976). The sugar hydroxysulfonates,
though not as stable as the carbonyl hydroxysulfonates, would still be stable in
the stomach, although dissociation in the small intestine would be predicted.
5 . Reactions of Sulfites with Proteins and Amino Acids
The disulfide bonds of cystine, peptides, and proteins can be cleaved by sulfite,
resulting in the formation of a thiol (R-SH) and an S-sulfonate (R-SSO,)
(Means and Feeney, 1971). The reaction goes essentially to completion with free
cystine at physiological pH, but the disulfide bonds of many proteins are unreac-
tive presumably because of steric hindrance or an unfavorable electronic environ-
ment (Gunnison, 1981). Denatured proteins are more susceptible to this sul-
fitolysis reaction.
Sulfites also react with cysteinyl residues of proteins and peptides (Green,
1976; Schroeter, 1966). The reaction product with cysteine is P-carboxyethylthi-
osulfonate (Schroeter, 1966). The formation of amine bisulfites has also been
reported from the reaction of sulfites with tertiary amines and Schiff's bases
arising from the browning reaction (Joslyn and Braverman, 1954). These reac-
tions with amines and thiols would predominate in food systems such as flour
doughs where gluten is a major constituent (Thewlis and Wade, 1974).
Methionine can be oxidized to methionine sulfoxide by sulfites via a free
radical mechanism (Yang, 1970, 1984). Tryptophan can be destroyed by a

sulfite-inducedfree radial reaction (Yang, 1973, 1984). The relative stabilities of
the sulfite adducts with proteins and amino acids are unknown.
6 . Reactions of Sulfites with Vitamins
Sulfites can apparently react with a number of vitamins, including thiamine,

ascorbic acid, vitamin B,,, and vitamin K.
The sulfitolysis of thiamine has been known for many years and is considered
deleterious, since it destroys the nutritive value of this vitamin (Schroeter, 1966).
For this reason, sulfites cannot legally be added to foods, such as meat, that are
considered to be prime sources of thiamine in the United States. Such additions
are allowed in other countries. The sulfitolysis of thiamine is an irreversible
nucleophilic attack of sulfite on the quaternary nitrogen of the thiazole ring; the
products of the reaction are pyrimidine sulfonic acid and 4-methyl-5P-hydroxy-
ethylthiazole. The kinetics of this reaction are first order with respect to both
reactants, and the half-life of 10 p M thiamine in the presence of 1 mM sulfite is
about 13 hr (Gunnison, 1981).
Sulfites can also react with the nonenzymatic browning products of ascorbic
acid. Wedzicha and McWeeny (1974a,b) investigated the reaction products of
ascorbic acid, amino acids, and sulfite; a large portion of the total sulfite residues
obtained from such reactions could not be recovered efficiently by the acidic
distillation procedure of Monier-Williams (1927). They succeeded in identifying
3-deoxy-4-sulfopentosuloseand 3-deoxy-4-sulfohexosuloseas two extremely
stable sulfite adducts in the reaction mixtures and sulfited, dehydrated cabbage
(McWeeny, 1979; McWeeny et al., 1974; Wedzicha and McWeeny, 1974b). Up
to 80% of the SO, in dehydrated cabbage may be in the form of such stable
adducts (McWeeny, 1979). The sulfite adducts are extremly stable in vivo
(Walker et al., 1983a).
Cobalamins react readily with sulfites. Most of the studies involve hy-
droxycobalamin, a naturally occumng form of vitamin B ,2 (cyanocobalamin). A
complex is readily formed between hydroxycobalamin and sulfite (Kaczka et a l .,
1950). The reaction has a very favorable equilibrium constant (Firth et al.,
1969). Cobalamins can actually catalyze the oxidation of sulfite to sulfate
(Kaczka et al., 1950; Smith et al., 1952).
Sulfite adds reversibly to the 2,3 double bond of menadione (vitamin K3), a
water-soluble synthetic form of vitamin K (Shih and Petering, 1973). The sulfo-
nate adduct is probably dissociable in the body, since the adduct can serve as a
source of vitamin K when fed to animals (Nir et al., 1978). The reaction rate for
the naturally occumng fat-soluble forms of vitamin K (vitamins K, and K,) with
sulfite is unknown.
Sulfite also forms reversible adducts with the pyridine and flavin nucleotides.
Sulfite adds to the 3,4 double bond of nicotinamide adenine dinucleotide (NAD)
(Shih and Petering, 1973). The sulfite-NAD adduct is not particularly stable, but
its stability is enhanced if the NAD is associated with an enzyme (Gunnison,
1981). With the flavin nucleotides, flavin adenine dinucleotide (FAD) and flavin
mononucleotide (FMN), the addition reaction occurs at the N-5 atom of the
isoalloxazine ring (Muller and Massey, 1969). The flavin nucleotide-sulfite
adducts are even less stable than the NAD-sulfite adduct, but again the stability
is enhanced if they are bound to protein (Muller and Massey, 1969). The reac-
tions of sulfite with enzyme-bound NAD, FAD, and FMN result in inhibition of
the enzyme (Gunnison, 1981).
Bisulfite has been shown to form nucleophilic adducts with folate and di-
hydrofolate (Vonderschmitt et al., 1967). The reaction can occur at pH 6.5, but,
because of an unfavorable equilibrium, it requires a considerable excess of
bisulfite to obtain any appreciable quantity of adduct. The adduct is also unstable
in the presence of oxygen.
Sulfites can also destroy p-carotene, the precursor of vitamin A (Yang, 1984;
Wedzicha and Lamikanra, 1983). The mechanism apparently involves free radi-
cals. Sulfite-induced lipid peroxidation can initiate this reaction.
7. Reactions of Sulfites with Nucleic Acids and Nucleotides
A variety of reactions have been described between sulfite and nucleic acids or
nucleotides (Gunnison, 1981; Hayatsu, 1976). Certain of these reactions are
thought to be responsible for the mutagenicity of sulfites which is observed in
certain systems (see Section III,C,4).
Sulfites can add reversibly to the 5,6 double bonds of uracil and cytosine and
their derivatives (Hayatsu, 1976; Shapiro et al., 1970b, 1973). With uracil, the
reaction is most rapid at pH 7, is readily reversible at pHs above and below pH 7,
and is dependent on relatively high sulfite concentrations (Hayatsu et a l . , 1970;
Pitman and Jain, 1979; Shapiro et a l . , 1976). Uridine can be regenerated from
the sulfonate adduct by removal of free sulfite from the reaction mixture (Rork
and Pitman, 1974). The cytidine adducts are most stable at acid pH, and their
formation is also dependent on high concentrations of sulfite (Shapiro et a l . ,
1974). The cytidine adducts are unstable at physiological pH (Shapiro et a l . ,
1974). RNA and single-stranded DNA can be deaminated via this mechanism
(Shapiro et al., 1970a, 1973).
The 5,6-dihydrocytosine-6-sulfonatecan be deaminated under some condi-
tions to yield the uracil adduct (Shapiro et al., 1974). The deamination is cata-
lyzed by sulfite and occurs optimally at pH 5 (Shapiro e f al., 1974). At low
sulfite concentrations and physiological pH, the rate of this conversion is ex-
tremely slow (Slae and Shapiro, 1978). However, this conversion is thought to
be responsible for some of the observed mutagenic effects of sulfites (see Section
III,C,4).
Sulfite can also catalyze the transamination of cytosine and its derivatives with
primary and secondary amines to produce N4-substitutedcytosines (Shapiro and
Gazit, 1977). 5,6-Dihydrocytosine-6-sulfonate is an intermediate in this reaction
also. The reaction is slow and requires high concentrations of sulfite, but can
occur at physiological pH. Polycytidylic acid can also be involved in such
reactions.
Gunnison (198 1) suggests that sulfite-induced mutations may also be the result
of deamination of 5-methylcytosine to thymine.
In certain reaction mixtures, aerobic oxidation of sulfite to free radicals can
occur (Hayatsu, 1976). These free radicals can lead to a variety of reactions with
nucleic acids and their derivatives, including cleavage of the glycosidic linkages
of uridine and cytidine (Kitamura and Hayatsu, 1974), fission of the chains of
polyuridylic acid and polycytidylic acid (Kitamura and Hayatsu, 1974), breakage
of the phosphodiester bonds in DNA in phage T7 (Hayatsu and Miller, 1972),
and addition to the double bonds of 4-thiouracil and 6-isopentenyladenosine,
minor base constituents of yeast transfer RNAs (Stacey and Harris, 1963).
The reactions of sulfites with nucleic acids can modify the activities of these
biomolecules in in vitro systems. These modifications are detailed in the review
by Gunnison (1981). Shapiro and Braverman (1972) demonstrated that sulfite
adduct formation in poly(U) interferes with its ability to form helical complexes
with poly(A) and the ability of poly(U) to code for phenylalanine incorporation
into protein. Similar modification of messenger RNA from coliphage MS2 and
ribosomal RNA from Escherichia coli led to decreased incorporation of amino
acids into proteins (Braverman et al., 1975). Uracil-sulfonate adducts can also
interfere with the DNA polymerase reaction (Kai et al., 1974). Sulfite-induced
free radical reactions can inhibit the transforming activity of DNA from Bacillus
subtilis (Inoue et al., 1972), can inactivate bacteriophage A (Kudo et al., 1978),
and can cross-link the maturation and coat proteins with nucleic acids in RNA
bacteriophage MS2 (Turchinsky et al., 1974).
8. Reactions of Sulfites with Pigments
Anthocyanins and other phenols in wines can also react with sulfites (Bur-
roughs, 1975; Somers and Evans, 1977). Anthocyanin complexes with sulfites
are quite unstable, dissociating even under acidic conditions (Burroughs, 1975).
Therefore, the titrimetric methods for free SO, would be expected to give er-
roneously high results when applied to red wines (Burroughs, 1975). The antho-
cyanin-sulfite complexes would be expected to dissociate readily on exposure to
stomach acid.
9. Reactions of Sulfites with Fatty Acids
Sulfites in the concentration range of 0.5-10 mM can induce the oxidation of

unsaturated fatty acids in corn oil and free linolenic acid (Kaplan et al., 1975;
Lamikanra, 1982), presumably through a free radical mechanism. Sulfites also
enhanced lipid peroxidation in a rat liver homogenate (Inouye et al., 1980).
Southerland et al. (1982) showed that bisulfite could react directly with the
double bonds of mono- and polyunsaturated fatty acids. This reaction could be
inhibited by vitamin E and butylated hydroxytoluene.
10. Fate of Sulfites in Specific Foods
The proportion of added sulfite existing in the combined form would be

variable from food to food. In most foods, the combined forms of sulfite would
be expected to predominate. An exception would be sulfited lettuce, where
virtually all of the sulfite is present in the free form (Taylor et al., 1985). A
comparison of free, combined, and total SO, residue levels in several foods as
determined by the aeration-oxidation method is interesting (Mitsuhashi et al.,
1979). The percentage of the total SO, existing as free SO, is 2.3% for white
wine, 22.3% for concentrated orange juice, 14.8% for molasses, 34.4% for corn
starch, and 32.3% for frozen peeled shrimp. The remainder of the sulfite is in the
combined form.
The fate of sulfites in foods is thus an extremely complex situation. The
combination with organic constituents, the equilibrium between the various in-
organic forms, the volatilization of SO,, and the oxidation to sulfate can all be
important reactions. The relative importance of these reactions is dependent on
the particular food involved. Although many studies have reported residual SO,
levels in foods, a few studies with radiolabeled sulfites stand out as the only
systematic investigations of the fate of sulfites in foods.
Thewlis and Wade (1974) studied the fate of radiolabeled sulfites in hard
sweet biscuit doughs. The results indicated that 93% of the sulfite sulfur added to
the dough remained in the biscuit. About 30% of the sulfite was converted to
sulfate and another 73% appeared as combined sulfites, probably S-sulfopro-
teins. Less than 0.2% remained, as inorganic sulfite (free SO,) in the finished,
cooked biscuits.
Gilbert and McWeeny (1976) examined the fate of radiolabeled sulfites in
dehydrated vegetables (cabbage, carrots, and potatoes). .4lthough there were
some methodological problems with this study, between 35 and 45% of the
sulfite was free or dissociable sulfite in the dehydrated potatoes, while a small
percentage was converted to sulfate and the remainder was stable sulfite adducts.
The effects of storage, preparation, and cooking on the residual level in the
dehydrated products were not examined.
Further evaluation of the fate of [35S]sulfitein dehydrated cabbage and carrots

was conducted by Wedzicha et al. (1984). In cabbage, 28.9% of the added
sulfite remained as sulfite, and 8.7% was oxidized to sulfate. C-Sulfonates
accounted for 34.3%, while 29.0% was attributed to a combination of disulfites
hydroxysulfonates, and S-sulfonates. In carrots, only 3.0% of the added sulfite
remained as sulfite, while 10% was oxidized to sulfate. The higher level of
remaining sulfite in cabbage was attributed to the higher pH of blanching (pH 9-
10) for this product by comparison to carrots (pH 5). With carrots, C-sulfonates
accounted for 5.0% of the added sulfite, while 81.0% was attributed to the
combination of disulfites, hydroxysulfonates, and S-sulfonates. In dehydrated
cabbage, the number of combined sulfite products was large; eight radioactive
peaks were obtained by ion-exchange chromatography.
McWeeny et al. (1980) also studied the fate of sulfites during production of
strawberry jam from sulfited strawberries. The strawberries absorbed 78% of the
radiolabeled sulfite from the sulfite liquor, but 98.5% of this sulfite was either
reacted chemically or lost during jam production: 50% was boiled off (jam is
acidic, so volatilization of SO, is likely), 38% was converted to combined sulfite
adducts during storage of the strawberries, 11% was converted to combined
sulfite adducts during jam making, and only 1.5% remained as inorganic sulfite
in the finished jam.
In a study of the fate of sulfites in dried fruit, McBean (1967) showed that
much of the SO, was lost on drying and that of the remaining sulfite, 80-90%,
was in the combined form. Further losses of sulfite occurred during storage.
In shrimp, most of the residual sulfite is located on the shell (Weingartner et
al., 1977). That sulfite is mostly removed with the melting ice water, and further
reductions are accomplished with rinsing in water or hypochlorite solutions
(Weingartner et al., 1977). With 1.25% bisulfite dips, only 11.7% of the sulfite
residue on the shell remained after 15 days of storage on ice at 2"C, while no
residual sulfite was noted in the muscle following 10 days of storage
(Weingartner et al., 1977).
11. Critical Factors in Determination of the Fate of Sulfites in Foods
The first critical step in determination of the fate of sulfites in foods is the
absorption of the sulfites from dip solutions or SO, from the atmosphere into the
product. With dried fruits, the concentration of absorbed SO, is a function of the
sulfite concentration in the dip solution, the immersion time, and the pH (Staf-
ford et al., 1972). SO, absorption is higher at pH 2.5 than at pH 4.5 (Stafford et
al., 1972). With table grapes, sulfitation is accomplished by release of SO, from
in-package generators during transport and storage (Nelson, 1983; Nelson and
Ahmedullah, 1973, 1976). The released SO, in the atmosphere of the container
disappears within 3 weeks (Nelson and Ahmedullah, 1976). The level of sulfites
in the grapes is of course proportional to the amount of SO, exposure and varies
from 17 to 40 ppm, depending on the temperature treatment and package type
(Nelson and Ahmedullah, 1973).
The second important factor in determining the fate of sulfites in foods is the
nature of the processing treatments. As can be seen from the studies already cited
(Gilbert and McWeeny, 1976; McWeeny et al., 1980; Thewlis and Wade, 1974;
Wedzicha et al., 1984), sulfite levels can be altered in a number of ways: (1) The
sulfites can be physically lost as SO, if the pH of the product drops below pH
4.0, especially if the product is heated; (2) much of the sulfites in nonacid
products can be converted into combined sulfite adducts, many of which remain
to be characterized; (3) some of this combined sulfite will be in the form of
extremely stable products, which cannot be recovered by conventional methods,
so it will be “lost” as far as analysis is concerned; and (4)oxidation of sulfite to
sulfate can occur in some foods and may be particularly significant in wines and
flour doughs, perhaps because it is catalyzed enzymatically in these foods.
Leaching of sulfite brine solutions is also an important step in lowering sulfite
residuals in many products. An example would be maraschino cherries.
The third step in determining the fate of sulfites in foods is the effect of storage
on residual sulfite levels. Storage almost always diminishes the amount of in-
organic sulfite or free SO, in the product. In bottled red wine, free SO, is lost on
storage; the loss is correlated with the oxygen content of the wine and presum-
ably represents conversion to sulfate (Jacobs, 1976). In dehydrated potatoes, 46-
68% losses in residual sulfite were noted on 24 weeks of storage at 75”F,
depending on the type of storage container (Lisberg and Chen, 1973). In dried
apricots, loss of SO, is also dependent on the type of package, with greater losses
from air-permeable packages than air-impermeable packages (Davis et al.,
1973). In oxygen-permeablepackages, some of the loss was attributed to sulfate
formation (Davis et al., 1973). Between 50 and 80% of the total SO, residue is
lost from dried apricots in 48 weeks of storage at 25°C (Davis et al., 1973). In
dried apples, the loss of residual SO, was shown to be a function of storage
temperature (Sayavedra and Montgomery, 1983). At 1”C, virtually no SO, was
lost in 400 days of storage, while at 38”C, 80% of the residual SO, was lost
within 400 days (Sayavedra and Montgomery, 1983).
The final step in determining the fate of sulfites in foods is the effect of
preparation on residual sulfite levels. This final step has received little attention
until the recent concern over sulfites and virtually nothing has been published on
the subject. The cooking of sulfited Thai noodles diminishes the total SO, level
by about 70% (Kingkate et al., 1981).
Apparently, processing, storage, and preparation act largely to lower the lev-
els of residual sulfite in foods. The actual amounts of free and total sulfite
available at the point of consumption have received little study, but it is probable
that the lowest free sulfite concentrations would exist at that point. If, as we
30 STEVE L . TAYLOR ET AL.
propose in Sections 111 and IV, the inorganic sulfites are more toxic than com-
bined sulfites and are responsible for the asthmatic reactions, then this situation
is very beneficial. Much more study of the effects of storage and preparation on
residual sulfite levels will be necessary, along with confirmation of our hypoth-
esis that combined sulfite adducts are not triggers of the asthmatic response.
G . TREATMENT LEVELS VERSUS RESIDUAL LEVELS
Treatment levels bear virtually no resemblance to residual levels for the sul-
fites. As we have noted, numerous factors and reactions can affect residual levels
of the sulfites. The fate of sulfites in foods would be different for each food and
each set of treatment conditions. Subsequent storage and preparation conditions
would also affect residual levels.
Much is heard about the losses in SO, that occur during processing, storage,
and preparation. As we have described in the previous section, true losses of
sulfite as SO, can occur in foods with pHs below pH 4.The pH-dependent losses
of SO, can be influenced by time, temperature, humidity, light, and other
factors. True losses of SO, can also occur through physical separation processes
such as leaching. Losses of sulfite by oxidation to sulfate will occur in many
foods, but seem to be most important in fermented foods where conversion back
to sulfite is always possible. However, in the majority of cases, most of the lost
SO, is really not lost from the product at all. It is merely converted into stable
combined sulfites that will not release the SO, under the usual assay conditions.
These losses may be important in terms of converting the sulfites into forms that
are less likely to initiate an asthmatic or toxic response. Thus, the substantial
losses in inorganic sulfites that occur between treatment and consumption may be
more important in terms of lowering the hazard of sulfites in foods.
In the future, emphasis should be placed on assessment of consumption levels
rather than treatment levels. Further knowledge of the role of different forms of
sulfites will be necessary before we will know exactly what we should be mea-
suring, however (see Section 11,E,4).
H. EXPOSURE ASSESSMENTS
I. Consumer Exposure to Su@tes from Foods

The residue data available for assessing consumer exposure to sulfites in foods
are woefully inadequate. We have already detailed our misgivings about the
methods of measurement, the reliance on total instead of free and combined SO,,
and the lack of information on the effects of storage and preparation on residue
levels.
The Codex Alimentarius Commission (1975) has established a potential daily
intake for sulfiting agents as SO, of 2.1 mg/kg or 126 mg for a 60-kg person.
This level would represent the upper limit of sulfite intake in the population. The
Expert Panel on Food Safety & Nutrition of the Institute of Food Technologists
(1975) agreed with this estimate of the upper limit of intake. However, they
noted that wide variations in sulfite intake would occur in the population. The
panel concluded that most Americans consume no more than 10- 15 mg total SO,
per day or about 0.2 mg/kg for a 60-kg man. The Ad Hoc Review Group on the
Reexamination of the GRAS Status of Sulfiting Agents (Life Sciences Research
Office, 1985) estimated total sulfite intake of 10 mg/capita/day from the con-
sumption of food, wine, and beer, with food accounting for perhaps 6 mg of this
total. They estimated that the 99th percentile intake for total SO, does not exceed
180 mg/capita/day. While these estimates may be reasonable, we believe that,
based on our misgivings about the sulfite residue data, a continuing evaluation of
the levels of consumer exposure to sulfites would be desirable. In particular, it
would be interesting to have data on consumer exposure to inorganic and com-
bined sulfite residues in addition to data on total SO, residues.
Obviously, some foods will contribute more heavily to consumer sulfite ex-
posure than others. Many sulfited foods have rather low residue levels. Dried
fruits, dehydrated vegetables, dehydrated potatoes, wine, and sulfited restaurant
salads and potatoes will contribute higher levels of sulfites than most other
products. If these foods are used routinely in the diet, the 10- to 15-mg estimates
could easily be exceeded. In a hypothetical meal with 250 ml of wine containing
150 pprn total SO,, a restaurant salad (100-g serving) containing 600 ppm total
SO,, and dried apricots (25-g serving) containing 2500 ppm total SO,, a total
SO, intake of 160 mg would be achieved.
The joint FAO/WHO Expert Committee on Food Additives (1974) established
an acceptable daily intake (ADI) for sulfiting agents as SO, of 0.7 mg/kg or 42
mg for a 60-kg man. The Committee used animal toxicity data to arrive at this
AD1 (see Section 111,C). The AD1 could be exceeded by one sulfited restaurant
salad, several dried apricots, or 250 ml of wine having 175 ppm SO,. The 99th
percentile of total SO, intake (180 mg/capita/day) is equivalent to 3 mg/kg,
although the average per capita intake of total SO, of 10 mg/day equates to only
0.17 mg/kg (Life Sciences Research Office, 1985).
2. Consumer Exposure to Sulfites from Other Sources
The major alternate sources of sulfites and SO, are drugs and the atmosphere.
The use of sulfites in pharmaceuticals has been reviewed by Schroeter (1966).
Their use dates back to at least 1940 and probably earlier. They are used in drugs
primarily as antioxidants. Sulfiting agents have been used in sympathomimetic
amines, sulfonamides, antibiotics, steroids, vitamins, dextrose solutions, eye
medications, antisyphilitic drugs, indigocarmine solutions, anticoagulants,

heparin, phenothiazine compounds, bronchodilators, local anesthetics, and mor-
phine (Schroeter, 1966). The sulfites are commonly, but not universally, used in
such drugs. The levels of use of sulfites in drug formulations have not been
strictly controlled. They have historically been used at the minimum concentra-
tions necessary to provide adequate antioxidant protection. The concentration of
sulfites in the majority of the drug formulations has been in the range of 0.01-
1.O%, although a few may contain higher levels (Life Sciences Research Office,
1985; Schroeter, 1966). Exposure via drugs can be high but would be sporadic
for most individuals. However, some patients would have continual exposure to
sulfited drugs over an extended period of time. With drugs, the routes of ex-
posure may be respiratory and intravenous as well as oral.
SO, is evolved into the atmosphere from volcanoes and industrial processes.
Estimates are that 146 million tons of SO, are emitted annually into the atmo-
sphere from industrial processes and motor vehicles (Stem et al., 1973). The
chief sources are coal and oil burning and production of sulfuric acid. Estimates
are that another 1.5 million tons of SO, are released into the atmosphere each
year from volcanic activity, although considerable variations in volcanic activity
occur. National standards require that the average level of SO, in the atmosphere
over a 24-hr period not exceed 0.14 ppm on more than one day each year and that
the average annual atmospheric concentration not exceed 0.03 ppm. Daytime
SO, levels in industrialized areas could be expected to exceed 0.14 ppm on
occasion. Workplace environments can also be contaminated with SO,, with
several parts per million occurring at times. The threshold limit value (TLV) for
SO, in the workplace is 2 ppm averaged over an 8-hr day (American Conference
of Government Industrial Hygienists, 1982). Oxidation of SO, in the atmosphere
to sulfuric acid occurs and is the focus of concerns regarding acid rain. The rate
of oxidation is faster at higher humidity levels and may be as great as OS%/min
in fogs. Exposure to atmospheric SO, occurs via the respiratory route rather than
the oral route.
The contributions of drug sulfite residues and atmospheric sulfur oxides to the
total intake of sulfite have not generally been included in exposure assessments
for food additives. Estimates of daily exposure to SO, can be derived from the
0. I4-ppm, 0.03-ppm, and 2-ppm limits described above. Respectively, such
exposures would provide total SO, exposures of 3.8 mg/24 hr, 0.85 mg/24 hr,
and 24 mg/8 hr (Life Sciences Research Office, 1985).
Ill. SAFETY OF SULFITES IN FOODS

A. METABOLISM OF SULFITES
The key to the understanding of sulfite toxicity may lie in elucidation of sulfite
metabolism. Several researchers have proposed that defects in sulfite metabolism
among certain segments of the human population may put them at greater risk to
the possible toxic effects of sulfite ingestion (Calabrese et al., 1981; Jacobsen et
al., 1984). Certainly defective sulfite metabolism in experimental animals can be
correlated with enhanced sulfite toxicity (Cohen et al., 1973). Considerable
information is available on the metabolism of free inorganic sulfites. Unfortu-
nately, much less information is known concerning the metabolism of combined
sulfites.
I. Free Sulfites
Free sulfite is metabolized principally by sulfite oxidase, more precisely
known as su1fite:cytochrome-c oxidoreductase or sulfite:O, oxidoreductase (EC
1.8.3.1). The product of this oxidative reaction is sulfate, which can be rapidly
excreted in the urine. Sulfite oxidase is present in all animal species that have
been examined, although species variations are observed in the levels of activity.
The rat possesses the highest level of sulfite oxidase activity, having 3-5 times
greater activity than rabbits or rhesus monkeys (Gunnison et al., 1977) and
approximately 10-20 times greater activity than humans (Johnson and Ra-
jagopalan, 1976a,b). Sulfite oxidase can be found in most mammalian tissues
except blood, with the highest activities found in the liver, followed by the
kidney (MacLeod et al., 1961; Johnson et al., 1977). The enzyme is localized
subcellularly in the intermembranous spaces of the mitochondria (Gunnison,
1981; Kessler and Rajagopalan, 1972).
Sulfite oxidase has been purified from several sources (Cohen and Fridovich,
1971a; Johnson and Rajagopalan, 1976a; Kessler and Rajagopalan, 1972) and
studied extensively. The enzyme has a molecular weight of 115,000- 120,000,
depending on the species, comprised of subunits of approximately 55,000 MW
each. Sulfite oxidase is a molybdoprotein (Cohen et al., 1971; Kessler and
Rajagopalan, 1972) that also possesses a heme molecule in addition to the
apoenzyme (Cohen and Fridovich, 1971b). The reaction mechanism involves the
transfer of electrons from sulfite to the Mo6+ site in the enzyme. The electrons
are then transferred to the heme moiety and from there to cytochrome c, a
constituent of the mitochondria1 respiratory chain. This leads to the ultimate
production of sulfate and one molecule of ATP, with the reduction of 4 0, to
H,O.
Sulfite oxidase appears to be the major metabolic pathway for both endoge-
nous and exogenous sulfite (Gunnison, 1981). Endogenous sulfite arises from
the metabolism of the sulfur-containing amino acids, cysteine and methionine.
The conversion of sulfite to sulfate via sulfite oxidase is the final step in the
catabolism of these amino acids.
Endogenously produced sulfite cannot normally be detected in blood or other
tissues due to its rapid oxidation to sulfate and excretion in the urine (Gunnison et
al., 1981b). It has been estimated that humans excrete about 25 mmol(2400 mg)
in their urine each day, the majority (up to 24 mmol) of which is generated from
endogenous sulfite (Institute of Food Technologists Expert Panel on Food Safety
and Nutrition, 1975). Rats generate about 0.5 mmol sulfite/kg/day based on
urinary sulfate excretion in animals in sulfur balance (Whiting and Draper,
1980). Apparently, sulfite oxidase was evolved to protect animals from endoge-
nously produced sulfite (Gunnison, 1981). This same enzyme serves to metabo-
lize exogenous sulfite as well.
The capacity of sulfite oxidase is very high in mammalian species (Gunnison,
198 1; Institute of Food Technologists Expert Panel on Food Safety and Nutrition,
1975). Based on projections from in virro assays of sulfite oxidase, Cohen et al.
(1973) calculated that the enzyme could theoretically oxidize sulfite at a rate of
750 mmol/kg/day (48 g of SO,/kg/day). Using perfused dog livers, Wilkins et al.
(1968) demonstrated that sulfite could be oxidized at a rate of 0.8 mmol/kg/hr,
which equates to a daily rate of 19 mmol/kg (1 200 mg of SO,/kg/day). Oshino and
Chance (1975) showed that perfused rat livers were capable of even faster sulfite
oxidation, with a rate of 2.4 mmol/kg/hr or 58 mmol/kg/day (3700 mg of SO,/
kg/day). In experiments with intact animals, Yokoyama et al. (1971) and Bhagat
and Lockett (1960) observed that dog and rats, respectively, could metabolize
inhaled SO, and ingested bisulfite to sulfate readily, with the majority of the dose
appearing in the urine as sulfate within a short time after administration. Gibson
and Strong (1973) observed that the majority of an oral dose of sulfite equivalent to
50 mg SO,/kg was excreted in the urine as sulfate within 24 hr. They could not
detect urinary sulfite, indicating extremely efficient oxidative metabolism. Sulfite
is absorbed very rapidly from the gastrointestinaltract (Bhagat and Lockett, 1960;
Gibson and Strong, 1973), so only a small portion of any oral dose is excreted in
the feces (Gibson and Strong, 1973). Gunnison and Palmes (1976) and Gunnison
et al. ( 1 977) evaluated the rate of metabolism of intravenous doses of sulfite in
rabbits, rats, and rhesus monkeys. They observed that large intravenous doses of
sulfite could be oxidized to sulfate within minutes. The biological half-lives for
plasma sulfite were calculated to be 1-2, 3-4, and 10 min in rats, rabbits, and
rhesus monkeys, respectively, after intravenousdoses of 0.3-0.6 mmol/kg (Gun-
nison et al., 1977). The half-lives of sulfite increased somewhat with increasing
doses of sulfite, probably because of feedback inhibition of sulfite oxidase by
sulfate (Gunnison et al., 1977). These studies collectively indicate that animals
possess sufficient sulfite oxidase to handle both endogenously produced sulfite
and rather substantial amounts of exogenous sulfite in addition.
Perhaps one precautionary note should be added regarding the capacity of
sulfite oxidase. Oshino and Chance (1975) demonstrated with perfused rat livers
that the rate of hepatic sulfite oxidation is limited by diffusion. Only 25-40% of
the infused sulfite dose was removed from the perfusate by the liver on each
pass. The rates of hepatic sulfite oxidation were similar for normal livers and
livers from rats with diminished sulfite oxidase levels when sulfite was infused
slowly. Therefore, the diffusion-limited metabolism of sulfite would indicate
that, despite the capacity of hepatic sulfite oxidase, some sulfite will pass
through the liver unmetabolized. The significance of this finding is uncertain.
Obviously, extrahepatic tissues possess considerable sulfite oxidase (MacLeod et
af., 1961; Johnson et af., 1977). The studies with intact animals would suggest
that sulfite is metabolized rapidly in spite of this diffusion limitation. However,
in sulfite-sensitive asthmatics, this limitation means that some absorbed sulfite
would be expected to pass through the liver and reach other tissues such as the
lungs.
Free plasma sulfite originating from either endogenous or exogenous sources
is not detectable in normal rats, mice, or rabbits, but can be detected in rhesus
monkeys challenged with 2 mmol sulfite/kg/day in the drinking water (Gunnison
and Palmes, 1973, 1976). Gunnison et af. (1981a) were able to measure up to
70-800 p M sulfite in the plasma of normal and sulfite oxidase-deficient rats after
a gastric challenge with 2-9 mmol sulfitelkg. Free plasma sulfite has also been
observed in a child with severe sulfite oxidase deficiency (Shih et af., 1977).
Several other pathways exist for the metabolism of sulfite in addition to the
sulfite oxidase pathway. The minor pathways are metabolism to thiosulfate and
formation of S-sulfonate compounds. Thiosulfate is produced from the reaction
of sulfite with 3-mercaptopyruvate (3-MP), a reaction catalyzed by 3-mercap-
topyruvate sulfurtransferase (EC 2.8.1.2) (Westley, 1980). 3-MP arises from
cysteine catabolism, as does sulfite. Thiosulfate is detected only at very low
levels in the urine of normal humans or rats (Gunnison, 1981; Gunnison et af.,
1981b), but is excreted in large amounts among sulfite oxidase-deficient indi-
viduals of both species (Gunnison et af.,1981b; Shih et af.,1977). Thiosulfate is
somewhat unstable in urine and is likely excreted at greater rates than those
measured (Gunnison et af., 1981b). However, urinary excretion of thiosulfate
was unchanged from normal in the siblings of sulfite oxidase-deficient patients
(Irreverre et af., 1967), indicating that thiosulfate excretion is probably not
affected by heterozygous deficiencies of sulfite oxidase.
S-Sulfonate compounds are formed nonenzymatically by the reaction of sulfite
with the disulfide bonds of proteins, cysteine, and glutathione (Cecil, 1963).
Urinary cysteine S-sulfonate cannot be detected in normal humans or rats, but
has been detected in individuals with sulfite oxidase deficiency in both species
(Gunnison et al., 1981b; Johnson et al., 1980; Shih ef al., 1977). However, only
the S-sulfonates with low molecular weights would be excreted in the urine, so
such measurements may not be totally indicative of S-sulfonate formation. Since
S-sulfonates can be formed in the extracellular compartments, this pathway may
have some significance in the disposition of exogenous sulfite, even with the
existence of high levels of sulfite oxidase. S-Sulfonates have been found in the
plasma of rabbits and rhesus monkeys after exposure to sulfite by ingestion or
injection and to SO, by inhalation (Gunnison and Palmes, 1973, 1974, 1978).
Plasma S-sulfonates are not detectable in rats unless the gastrointestinal tract is
bypassed by parenteral administration of sulfite (Gunnison and Palmes, 1978).
This has led to speculation that S-sulfonate formation is greater in animals with
comparatively lower sulfite oxidase levels (Gunnison et al., 1977). The high
activity of sulfite oxidase in rat small intestine (Johnson et al., 1974) may
prevent plasma S-sulfonate formation in that species. Protein S-sulfonate com-
pounds are very stable in vivo by comparison to sulfite; their biological half-lives
can be 1-3 days (Gunnison and Farruggella, 1979; Gunnison and Palmes, 1973,
1978). The mechanism for clearance of protein S-sulfonates from the body is not
known.
A better evaluation of the comparative importance of the sulfate, thiosulfate,
and S-sulfonate pathways for excretion of ingested and endogenously produced
sulfite can be obtained by comparing the urinary excretion rates. In normal
humans, urinary S-sulfonate cannot be detected (Shih et al., 1977; Johnson et
al., 1980). Humans have been observed to excrete thiosulfate at a rate of 32 k 13
pmo1/24 hr (Sorbo and Ohman, 1978). Urinary excretion of sulfate in humans
can reach 25,000 kmo1/24 hr (Institute of Food Technologists Expert Panel on
Food Safety and Nutrition, 1976), although some of this undoubtedly represents
ingestion of sulfate as such.
Obviously, normal individuals have a tremendous capacity to metabolize sul-
fite by several different pathways, with the sulfite oxidase pathway being the
most important. However, profound sulfite oxidase deficiency has been reported
on several occasions in humans (Duran et al., 1979; Irreverre et al., 1967; Mudd
et al., 1967; Ogier et al., 1982; Shih et al., 1977). The deficiency is charac-
terized by increased urinary excretion of sulfite, thiosulfate, and cysteine S-
sulfonate and decreased excretion of sulfate. The deficiency is congenital and
may be due to defects in the apoenzyme or in the metabolism of the essential
molybdenum cofactor. The individuals with profound sulfite oxidase deficiency
exhibit dislocated ocular lenses and severe neurological abnormalities resulting
in mental and physical retardation. In at least one case, death occurred at an early
age (Institute of Food Technologists Expert Panel on Food Safety and Nutrition,
1975). The levels of sulfite oxidase in humans can be determined using cultured
skin fibroblasts (Shih et al., 1977). Using this technique, Shih et al. (1977)
identified the parents of a child with sulfite oxidase deficiency as probable
heterozygotes by virtue of their intermediate levels of the enzyme. Recently,
Jacobsen et al. (1984) have demonstrated that sulfite-sensitive asthmatics may
also have heterozygous levels of sulfite oxidase activity.
Sulfite oxidase deficiency can be produced in rats by feeding diets high in
tungsten (W) relative to molybdenum (Mo) (Johnson et al., 1974). The W
competes with Mo for the Mo-binding site on sulfite oxidase. The loss of func-
tional sulfite oxidase is slow; Gunnison et al. ( I98 1b) observed a half-life of 4
days for hepatic sulfite oxidase at a W:Mo ratio of 5800. Eventually with pro-
longed administration of high W:Mo diets, a steady-state level of sulfite oxidase
activity is reached at about 1% of the normal adult level for the W:Mo ratio of
5800 (Gunnison et al., 1981b). The amount of activity at the steady-state level is
a function of the W:Mo ratio (Gunnison et al., 1981b). Gunnison (1981) has
argued convincingly that the sulfite oxidase-deficient rat could serve as a model
for sulfite metabolism and toxicity studies in humans, since the activity of the
enzyme can be adjusted via the W:Mo ratio of the diet to approximate various
human conditions: normal, heterozygous deficient, and deficient. The normal rat
possesses considerably more sulfite oxidase activity than the human (Johnson
and Rajagopalan, 1976a,b), making it a poor choice for an animal model. The
sulfite oxidase-deficient rat displays increased plasma sulfite levels after an
exogenous challenge of sulfite and increased urinary excretion of thiosulfate and
S-sulfonate (Gunnison et al., 1981b). In the absence of an exogenous sulfite
challenge, elevated plasma sulfite is not observed in these rats until the sulfite
oxidase activity drops to I-2% of normal rat levels, a testament to the efficiency
of endogenous sulfite oxidation (Gunnison et al., 1981b). Apparently, a very
small amount of sulfite oxidase is needed to prevent the escape of endogenous
sulfite from the cell.
2 . Combined Sulfites
Very little information is available on the metabolism of the various combined

forms of sulfite. Intuitively, one would expect that some combined sulfites
would be metabolized in a manner similar to inorganic sulfites. An example
would be glucose hydroxysulfonate; the pH stability of glucose hydroxysulfonate
would seem to indicate hydrolysis to the free sulfite under the neutral pH condi-
tions of the small intestine. However, this probable instability of the reducing
sugar hydroxysulfonates has not been established in vivo through experimenta-
tion.
Gibson and Strong (1974) demonstrated that sulfited proteins may be metabo-
lized to sulfate in a manner similar to inorganic sulfite. Oral administration of
35S-labeled sulfited rat serum protein to rats was followed by excretion of 40-
55% of the label in the urine within 24 hr. Most of the urinary label was sulfate.
Most of the remaining label was found in the feces; very little (7-17%) was
incorporated into body tissues. Sulfited proteins are apparently metabolized to
sulfate rather quickly. However, additional studies are needed to determine if
other sulfited proteins are metabolized in the same manner as the sulfited rat
semm proteins.
The metabolism of 3-deoxy-4-sulfohexosulose(DSH), a product of the reac-
tion between an intermediate in the Maillard browning reaction and sulfite

(Wedzicha and McWeeny, 1974a,b), has been recently studied in detail (Walker
et al., 1983a). [I4C]DSH was administered orally to mice and rats. In mice, 50%
of the dose was found in the feces, 13% in the cage washings, and 29% in the
urine. In rats, fecal excretion varied between 58.5 and 73%, with urinary excre-
tion between 16.5 and 31%. Carcass levels of label in both rats and mice were
less than 0.1% after 72 hr. The results indicate that DSH is poorly absorbed, but
once absorbed is rapidly metabolized and/or excreted. However, significant
transient levels of radioactivity were observed in liver, kidney, lung, and pan-
creas of both species and in rat testes and mouse bladder.
Much further work is needed to define the metabolism of the combined forms
of sulfite that predominate in foods.
B. HUMAN CHALLENGE TRIALS
Several human challenge trials have been conducted with sulfites and sulfited
beverages. Many of these human challenge trials were conducted in the early part
of this century; a good review of these studies is provided by Cluzan ef al.
(1965). In the earliest of these studies, Leuch (1895) challenged 30 volunteers
with a small amount of wine (30-50 ml) containing various quantities of SO,.
He observed that, with SO, quantities above 45 mg of free SO,, the subjects
complained of throat irritation, stomach bum, and headache. Walbaum (1906)
also reported gastrointestinal irritation in subjects who received much larger
amounts-310-620 mg of sodium sulfite at 4 times per day over a 4-day inter-
val. Wiley (1907) challenged 12 subjects over a 20-day period with increasing
amounts of SO, in aqueous solution (80-100 mg) or in the form of capsules of
sodium sulfite (1 15-760 mg). He reported some subjective complaints such as
headache, hearing impairment, and weakness, but also observed renal impair-
ment and hypochromic anemia. Rost and Franz (1913) challenged human volun-
teers with 1.0 g (17 mg/kg) of sodium sulfite per day and noted no gastroin-
testinal complaints, but vomiting occurred when the dosage was increased to 4-
5.8 g/day (70-100 mg/kg). This result was substantiated by Lafontaine and
Goblet (1953, who observed gastrointestinal irritation and vomiting in humans
when sulfite doses in excess of 3.5 mg/kg were administered. While these
experiments were interesting and established that very high doses of sulfite are
acutely toxic to humans, one must recognize that methods for sulfite analysis
may have been rather crude at the time of the early experiments and that the
prolonged trial of Wiley (1907) may have been compromised by the known
destruction of thiamine by sulfite. Wiley would not have known that thiamine
supplementation was necessary.
Perhaps the best human challenge trial was conducted by Hotzel er al. (1969).
They placed 12 volunteers on a normal diet for 15 days followed by a thiamine-
deficient diet for 15 days. Six subjects were then challenged with beverages that
provided 400 mg of SO, per day (50 mg as NaHSO, and 350 mg as sodium
glucose hydroxysulfonate) for 25 days; the other 6 subjects received the same
beverages containing no added SO, for 25 days. Sulfite administration was then
discontinued for 10 days, and the subjects were given oral thiamine supplementa-
tion for 2 days. Clinical examinations, enzyme activity measurements, hema-
tocrit values, and erythrocyte counts were taken throughout the experiment on all
volunteers. None of the subjects exhibited any abnormalities or changes that
could be attributed to sulfite. This study has been taken to indicate that sub-
chronic administration of sulfite to humans is without effect even when the
subjects are thiamine deficient (Life Sciences Research Office, 1976).
C. ANIMAL AND CELLULAR TOXICITY STUDIES
1 . Acute Toxicity
The free inorganic sulfites do not have a high degree of acute toxicity. Most of
the LD50 studies have involved intraperitoneal or intravenous routes of admin-
istration rather than the more relevant oral route of administration. A tabulation
of the LD,,s of the sulfiting agents is provided in Table VI. The general order of
acute toxicity is intravenous > intraperitoneal > per 0s. By sonie routes of
administration in some species, a dose killing 50% of the animals was not
achieved; these doses are reported in Table VI as LD,,,. The LD,, values do not
always agree when independent studies are compared such as the LD,,s for
intravenous administration of Na,SO, to mice (Table VI). Many factors could
explain the discrepancies. In particular, it must be remembered that sulfites are
unstable in aqueous solutions, so any storage of the solution before dosing would
result in a loss of sulfite and an apparent decrease in toxicity. Cohen et al. (1973)
determined that the intraperitoneal LD,, of NaHSO, was 181 mg/kg (1 11 mg
SO,/kg) in sulfite oxidase-deficient rats as compared to 473 mg/kg (291 mg SO,/
kg) in normal rats.
The acute toxicities of the combined forms of sulfite have received little study.
Lewis and Tatken (1979) list an LD,,, of 1220 mg/kg as SO, for oral admin-
istration of acetaldehyde hydroxysulfonate in the rabbit. Walker et al. (1983b)
could not determine an oral LD,, for DSH in rats or mice and conclude that the
oral LD,, for DSH exceeds 5 g/kg. These scattered results would tend to indicate
that some of the combined sulfites are less toxic than the inorganic sulfites, but
further studies are needed on additional compounds and on other routes of
TABLE VI
LD,, AND/OR LD,,, OF SULFITING AGENTS
LD5,, LD,,, SO2 equiv.

Species Route" Chemical (mg/kg) (mg/kg) (mglkg) Reference
Mouse iP NaHS03 675 416 Wilkins et al. (1968)

iv NaHS03 130 80 Hoppe and Goble (1951)
iv Na2S0 130 66 Lewis and Tatken (1979)
iv Na2S03 155 79 Jaulmes (1970)
iv Na2S03 175 89 Hoppe and Goble (195 I )
iP Na2S03 . 7 H 2 0 277 70 Nofre er al. (1963)
Rat PO SO26 1040 1040 Jaulmes ( 1970)
Po s02c 2000 2000 Jaulmes ( 1970)
iP NaHS03 650 400 Wilkins et al. (1968)
iP NaHS03 473 29 1 Cohen et al. (1973)
iv NaHS03 115 71 Hoppe and Goble (1 95 I )
iv Na2S03 I15 58 Lewis and Tatken (1979)
Po K2S205 1800 1037 Lauteaume er al. ( 1 969)
Parenteral Na2S205 500 337 Ezrielev (1968)
Rabbit iP NaHS03 300 I85 Wilkins et al. (1968)
iv NaHS03 65 40 Hoppe and Goble ( I 95 1)
Po Na2S03 - 1435 Lewis and Tatken ( 1 979)
sc Na2S03 - I52 Lewis and Tatken (1979)
iv Na2 s 2 O5
- 129 Lewis and Tatken (1979)
Hamster iv NaHS03 95 58 Hoppe and Goble (1951)
Guinea pig iv Na2S03 - 102 Lewis and Tatken (1979)
sc Na2S03 - 305 Lewis and Tatken (1979)
Cat iv Na2S03 - 66 1 Lewis and Tatken (1979)
Dog iP NaHS03 244 I50 Wilkins et a/. (1968)
Human iv Na2S03 . 7 H 2 0 - 189 Lewis and Tatken (1979)
ip, Intraperitoneal; iv, intravenous; PO, per 0s; sc, subcutaneous.

As a 6.5% aqueous solution.
As a 3.5% aqueous solution.
administration before firm conclusions can be drawn. Walker (1984) notes that
much less information is available on the toxicity of the combined sulfites than is
known about the reactions leading to their formation.
2 . Subchronic and Chronic Toxicity
Numerous subchronic and chronic toxicity studies have been conducted on the
free inorganic sulfites. For the purposes of this review, the early studies will be
ignored because of the distinct possibility that many of the toxic manifestations
were the result of thiamine deficiency, since the impact of sulfite on thiamine
was not recognized at that time. Some of these studies have been reviewed
elsewhere (Cluzan et al., 1965; Ti1 et al., 1972a).
The more recent studies of subchronic and chronic toxicity of sulfites gener-
ally fall into two categories: those in which the sulfite was administered with the
drinking water and those in which the sulfite was administered with the diet.
Both of these approaches have disadvantages. Sulfites are unstable in drinking
water; Lockett and Natoff (1960) observed a 20% decline in sulfite levels within
48 hr. Some investigators have ignored the stability problems, making their
studies difficult to interpret. The drinking water approach has been favored by
some investigators because it avoids the problem of thiamine destruction that is
inherent with the incorporation of sulfites into the diet. Gunnison et al. 11981a)
showed that sulfites do not destroy thiamine systemically, although Gunnison
(1981) notes that sulfite ingested with drinking water might destroy some
thiamine in the stomach. The incorporation of sulfites into the diet is also fraught
with difficulties, since the sulfites are extremely reactive with other dietary
components. These reactions can substantially decrease the free sulfite content of
the diet and makes interpretation of the results difficult.
Many of the recent studies have focused on attempts to confirm the finding
reported by Fitzhugh et al. (1946), who administered NaHSO, to rats in their diets
for up to 1 year. The diet was often left in the feeder cups unchanged for up to 1
week, which resulted in losses through reaction of up to 75% of the sulfite
(Gunnison, 1981). The diets contained 0.05-2.0% NaHSO, (0.08-13
mmol/kg/day) originally. Fitzhugh et al. (1946) noted toxic manifestations at
bisulfite levels above 0.1 % that included growth retardation, clinical polyneuritis,
“spectacle” eyes, bleached incisor teeth, brown uteri, atrophy of various viscera,
calcified renal tubular casts, atrophy of bone marrow and bone, myocardial
necrosis and fibrosis, and gastric squamous epithelial hyperplasia. These results
have been questioned because of the diminishing levels of sulfite in the diets and
the probable destruction of thiamine in the diet. Fitzhugh et al. (1946) attempted to
correct the thiamine deficiency through supplementation. Polyneuritis was not
observed in the supplemented animals, but the other toxic manifestations per-
sisted. Gunnison (198 1) has questioned whether the thiamine supplementation
was sufficient to entirely correct the deficiency. Based on the severity of the
manifestations observed in this experiment by comparison to others (see later), we
would echo these sentiments and further note that other dietary factors might have
been affected by the storage of diet in the feeder cups for prolonged periods which
could lead to other deficiencies. Bhagat and Lockett (1964) noted that diet
prepared with metabisulfite and stored at room temperature would quickly become
deficient in thiamine. On prolonged storage of 3-4 months at room temperature,
the diets would cause problems, such as chronic diarrhea, that could not be
reversed by thiamine supplementation (Bhagat and Lockett, 1964). This is an

indication that other factors in the diet may also be destroyed by sulfite addition
and contribute to the toxicological evaluations if diets are not properly prepared
and stored.
The results of Fitzhugh et al. (1946) have not been corroborated in other
chronic toxicity studies. Three of these studies have involved the incorporation
of sulfites into the drinking water (Cluzan et al., 1965; Lauteaume ef al., 1965;
Lockett and Natoff, 1960). Lockett and Natoff (1960) administered 0, 375, and
750 ppm of SO, as Na,S,O, in the drinking water of rats in a 3-year multigenera-
tion study. They observed no effects of sulfite on growth, food intake, fecal
output, fertility, weight of the newborn, growth during lactation, or any of the
pathological signs noted earlier by Fitzhugh et al. (1946). The study of Lockett
and Natoff (1960) was compromised by the losses of SO, in the drinking water
(10% in 24 hr) and the fact that many of their animals developed respiratory
ailments during the course of the experiment. Cluzan et al. (1965) conducted a
multigeneration study in rats over a 20-month period, administering 700 ppm of
SO, as K,S,O,. They found no evidence of toxicity as mortality, growth rate,
feed and water consumption, organ weights, hematological values, clinical
symptoms, and reproductive capacity were equivalent to controls. Cluzan et al.
(1965) did not provide any evidence for the stability of sulfites in their experi-
ment. Lauteaume et al. (1965) administered sulfites by gastric intubation to rats
over a 2-year period at a rate of 3 m1/100 g body weight/day. The rats were
divided into three groups that received (1) water and 450 ppm SO,, (2) red wine
with 110 pprn SO,, or (3) red wine with 450 ppm SO,. The sulfites did not affect
growth rates, reproduction, or the development of macroscopic or microscopic
lesions.
The most thorough evaluations of the chronic toxicity of sulfites were per-
formed by Ti1 et al. (1972a,b) using incorporation of Na,S,O, into the diets of
rats and pigs. Losses of sulfite through reactions with other dietary components
were minimized by frequent diet preparation and frozen storage. The amount of
sulfite loss was measured and the data were reported using the corrected values.
Thiamine was added to the diets 'in sufficient quantities to overcome any
thiamine destruction by sulfite. On a percentage basis, sulfite losses were great-
est at low sulfite concentrations, while thiamine losses were highest at high
sulfite concentrations. Sulfite losses ranged from 4.5 to 22%, while thiamine
losses ranged from 1.7 to 15.4%.
In the rat study (Ti1 ef al., 1972a), the added levels of Na,S,O, were 0.125,
0.25,0.50, 1.O, and 2.0%. The study was conducted over a period of 2 years and
involved three generations. The 2.0% Na,S,O, diet caused slight growth retar-
dation in the F, and F, generations, but had no effect on the F, generation. Part
of this effect is explained on the lower birth weights in the F, and F, generation,
although other reproductive effects were absent. Occult blood was observed in
the feces of rats receiving the 1.0% and 2.0% Na,S,O, diets. Kidney weights
were slightly increased with the 2% diet in the F, females only, and this change
was not accompanied by any functional or histopathological changes in the kid-
neys. Histopathological observations were largely normal except for the exis-
tence of hyperplasia in the fore and glandular stomachs of the rats receiving 1%
and 2% Na,S,O, diets. This hyperplasia was noted in all three generations and
was observed to a lesser extent in the forestomachs only of some rats on 0.5%
Na2S,0, diets. Beems et al. (1983) have further examined this hyperplastic
response and concluded that it involves chief cells, but the mechanism of the
response remains unknown. The no-effect level from the rat study was 0.25%
Na,S,O, in the diet, which is equivalent to 72 mg SO,/kg/day after conversion
and correction for sulfite losses. The Joint FAO/WHO Expert Committee on
Food Additives (1974) used the results of this experiment to establish the AD1 of
0.7 mg SO,/kg by simply applying a 100-fold safety factor to the no-effect level
obtained by Ti1 er al. (1972a).
Although this experiment is the most carefully controlled study of the chronic
toxicity of sulfites in existence, it has been criticized. Hickey er al. (1976) point
out that the levels of sulfite oxidase in humans are much lower than the levels in
normal rats, so a study of sulfite toxicity using ,normal rats is not justified.
Subchronic toxicity studies with sulfite oxidase-deficient rats clearly demonstrate
that such animals are more susceptible to the toxic effects of sulfites (Gunnison er
al., 1981b). However, the 100-fold safety factor is intended partly to correct for
such differences in detoxification pathways. The study of Ti1 et al. (1972a)
should be recognized as a study of the toxicity of total sulfite rather than free
sulfite, however. Ti1 er al. (1972a) analyzed for sulfite residues in their diets by
the method of Reith and Willems (1968), which detects total sulfite levels.
Therefore, some of the Na,S,O, added to the diet may have reacted with dietary
components but would be recovered as SO, during the analytical procedure. In
all likelihood, Ti1 er al. (1972a) underestimated the degree of free sulfite loss by
reaction with dietary components.
Ti1 et al. (1972b) also conducted a chronic toxicity study in pigs. The tech-
niques were identical to those used in the rat study (Ti1 et al., 1972a). A 48-week
feeding period was employed. The results varied somewhat, however. Some
growth retardation was noted in diets having 0.83 and 1.72% residual sulfite,
although this was due to diminished food intake, as a later paired feeding trial did
not demonstrate any differences in growth rates or food conversion. Organ to
body weight ratios were increased at the 0.83% and 1.72% levels for liver,
kidney, heart, and spleen, although this is ascribed to the lower body weights. In
contrast to the rat study, no occult blood was observed in the feces. Histo-
pathological examinations were normal except for mild inflammation and hyper-
plasia in the stomach at the 0.83% and 1.72% levels at both the 15-week and 48-
week observation periods.
Subchronic toxicity studies were also conducted by Ti1 et al. (1972a,b) on
both rats and pigs. In rats (Ti1 et al., 1972a), high sulfite levels (0-8%) were fed
in the diet for 10-56 days. Diets containing 6% sulfite caused marked growth
depression, reduced food intake, and lowered food conversion efficiency. Severe
anemia, increased spleen weights, and slightly elevated leukocyte counts were
also observed. The hyperplasia of the forestomach was found with 1% sulfite or
more, while glandular stomach hyperplasia, hemorrhagic erosions, necrosis, and
inflammation were found with 4% sulfite or more. Forestomach ulcers and
papillomatous elevations occurred at 6 and 8% sulfite. All of these effects were
reversible. In pigs (Ti1 e f al., 1972b), the changes observed after 15 weeks of
feeding were similar to those encountered after 48 weeks of feeding. Bhagat and
Lockett (1964) observed diminished growth rates in rats fed 0.6% Na,S,O, in
the diet over a 5- to 7-week period, but this effect could be reversed by supple-
mentation with thiamine. Gunnison et al. (198 la) confirmed the observation of
anemia in rats and attributed it to the interaction of sulfite with dietary factors,
perhaps vitamin B12. Gunnison et al. (1981a) conducted their experimeots with
sulfite oxidase-deficient rats and showed elevated excretion of S-sulfonates can
occur after administration of low levels of sulfite (0-3.5 mmol/kg/day). Ob-
viously, sulfite oxidase-deficient rats are more susceptible to the toxic effects of
oral sulfite, and Gunnison (1981) has suggested their use in sulfite toxicity
studies.
Few experiments have been conducted on the chronic and subchronic tox-
icities of combined forms of sulfite. Dietary studies such as those by Ti1 et al.
(1972a,b) are probably tests of the toxicity of some mixture of free and combined
sulfites. Gibson and Strong (1973) used glucose hydroxysulfonate in some of
their metabolism studies. Glucose hydroxysulfonate is likely to be stable to
stomach acid, but likely decomposes to free sulfite in the neutral pH of the small
intestine. They found no histological abnormalities in the livers and kidney of
rats dosed with glucose hydroxysulfonate for 30 days. Walker et al. (1983b) did
not observe any adverse effects after oral administration of DSH to rats for 14
days.
3. Carcinogenicity
Tumorigenic effects were not encountered in any of the chronic toxicity tests
described above. In addition, Tanaka et al. (1979) failed to find any tumors in a
carcinogenicity test of K,S20, in mice; 0, 1, and 2% K,S,OS was administered
in the drinking water. Gunnison et al. (1981a) noted a small incidence (4/ 149) of
mammary adenocarcinoma in sulfite oxidase-deficient rats as compared to O / 143
in controls after 5 months of feeding of tungsten, but the effect was not statis-
tically significant.
4. Mutagenicity
The mutagenicity of free inorganic sulfites has been extensively studied. The
subject has been reviewed in detail elsewhere (Gunnison, 1981; Shapiro, 1977),
and no attempt will be made here to provide such detail. The reactions of sulfite
with nucleic acids were covered in Section II,F,7. The mutagenicity of the
sulfites is thought to originate from the deamination of cytosine to uracil. The
involvement of sulfite-induced deamination of 5-methylcytosine to thymine in
the mutagenic process has also been considered, but the cytosine-to-uracil con-
versions are thought to be quantitatively more important (Wang and Ehrlich,
1980; Wang ef al., 1980).
Sulfites are capable of inducing mutations in vitro in several mutagenicity test
systems, including E. coli, y phage, T4 phage,yeast, and Vicia faba root mer-
istems (Chambers et al., 1973; Dorange and Dupuy, 1972; Hayatsu and Miura,
1970; Mukai et al., 1970; Njagi and Gopalan, 1982; Summers and Drake, 1971).
However, these experiments required high concentrations of sulfite and acid pHs
in the vicinity of pH 5 . When incubations were performed at neutral pH, no
measurable mutagenic response was observed (Mukai et al., 1970). MacRae and
Stich (1979) found that sulfite induces dose-related sister chromatid exchange in
Chinese hamster ovary cells, but the potency of this induction was relatively
weak. Sulfites can also cause chromosome damage when incubated in v i m with
oocytes from mice, cows, or sheep (Jagiello et al., 1975). However, they could
not induce chromosome aberrations in mouse oocytes cultured in vitro after an
intravenous injection of sulfite.
Despite the evidence for mutagenicity of sulfite in the systems described
above, there is no evidence for sulfite-induced mutagenesis in other systems. The
Food and Drug Administration contracted for mutagenicity studies in a variety of
systems, and the results of these tests are reported in the 1976 GRAS evaluation
document (Life Sciences Research Office, 1976). Sodium bisulfite was not mu-
tagenic in the host-mediated assay in mice, the dominant lethal assay in rats, the
in vivo cytogenetic assay in rats, and human tissue culture cells in vitro (Life
Sciences Research Office, 1976). Sodium sulfite and potassium metabisulfite
were not mutagenic in vitro in the Ames Salrnonellalmammalian microsome test
(Life Sciences Research Office, 1976). Sodium metabisulfite did cause mitotic
inhibition and damage to anaphase cells when added to human embryonic lung
cells in culture (Life Sciences Research Office, 1976). However, sodium meta-
bisulfite was not mutagenic in the host-mediated assay, the dominant lethal
assay, or in vivo cytogenetic assays (Life Sciences Research Office, 1976).
Generoso et al. (1978) showed that sulfite was negative in the dominant lethal
assay in mice after intraperitoneal injections. Drosophila ingesting a 0.08 M
solution of NaHSO, (5120 ppm SO,) displayed a mutation rate that was not
significantly different from controls (Valencia er al., 1972). Renner and Wever
(1983) were unable to induce cytogenetic damage as monitored by sister chro-
matid exchange, chromosome aberration, and the micronucleus test in sulfite
oxidase-deficient mice and Chinese hamsters after intragastric administration of
one or two doses of Na,S,O, (330 or 660 mg/kg) in aqueous solutions or fruit
juice. Bisulfite in aqueous solutions or in fruit or vegetable juices was not
mutagenic to Salmonella typhirnuriurn strains TA 1535, TA 1538, TA 100, or
TA 98, but an increase in revertants was obtained with strain his-G46 (Munzer,
1980). In this strain, more revertants were obtained with sulfited fruit or vegeta-
ble juices than aqueous solutions of sulfite (Munzer, 1980).
Some evidence also exists for a comutagenic effect of sulfites (Mallon and
Rossman, 1981). Enhanced ultraviolet mutagenicity was observed in Chinese
hamster V79 cells if they were exposed to 10 mM sulfite either during or
immediately following irradiation. A twofold increase in mutagenicity was ob-
served by comparison to irradiated controls not exposed to sulfite. With E. coli,
100 mM sulfite caused an eightfold increase in mutagenicity. Mallon and
Rossman (198 1) obtained evidence implying that sulfite was inhibiting excision
repair.
Sulfite can also be an antimutagen. Sulfite at 200 ppm is able to inhibit the
mutagenic effect of coffee in the Salmonellalmammalian microsome system and
the induction of prophage A (Suwa et al., 1982). Sulfite also suppressed the
mutagenicities of the 1,2-dicarbonyls, diacetyl and glyoxal (Suwa et al., 1982).
Almost no information is available on the mutagenicity of the various com-
bined forms of sulfite. Walker et ul. (1983b) demonstrated that DSH is not
mutagenic in the Ames Salmonellulmammalian microsome assay.
5. Teratogeniciry
Teratogenicity studies on NaHSO,, Na,S,O,, and K,S,O, have been con-

ducted in several species on behalf of the Food and Drug Administration; the
results of these evaluations were reviewed in the 1976 GRAS evaluation docu-
ment (Life Sciences Research Office, 1976). These sulfites were administered
orally to rats and mice on a daily basis on day 6 through day 15 of gestation and
similarly in hamsters except on day 6 through day 10 of gestation. The doses (in
mg/kg) for mice, rats, and hamsters ranged up to 150, 110, and 120, respec-
tively, for NaHSO,; up to 160, 110, and 120, respectively, for Na,S,O,; and up
to 125 and 155 in mice and rats, respectively, for K,S,O,. The incidence of
teratogenic effects was unchanged from control animals. Maternal and fetal
survival were also not affected by these sulfites.
Dulak et al. (1984) investigated the reproductive toxicology of sulfite in sulfite

oxidase-deficient rats. Exposure to sulfites from 3 weeks before mating until day
20 of gestation revealed no reproductive hazards for sulfite. Mating and pregnan-
cy rates, gestational weight gain, preimplantation loss, resorbed and dead
fetuses, litter size, fetal weights, and malformations were unaffected by sulfite
treatment.
6. Studies in Cell Cultures
Sulfites have a variety of effects on cultured cells. Sulfites are cytotoxic to

mouse fibroblasts, mouse liver cells, HeLa cells, Chorella pyrenoidosa cells,
and human lymphocytes in culture (Das and Runeckles, 1974; Thompson and
Pace, 1962; Timson, 1973). This cytotoxic effect was observed in the 0.1-20
mM range, although the minimum inhibitory concentrations varied among the
different cultures. DNA synthesis can be inhibited in chick embryo fibroblasts by
0.1-1 .O mM sulfite (Chin et al., 1977). Sulfite can also prevent the adhesion of
Chinese hamster cells to the substratum (Kudo et al., 1980). Kikigawa and
Iizuka (1972) showed that 7.5 mM sulfite inhibited the ADP- and collagen-
induced aggregation of rabbit platelets.
D. HYPERSENSITIVITY TO INGESTED SULFITES
1 . History of Asthma and Other Adverse Reactions to Sulfites
Recently, sulfiting agents have been reported to induce asthma when adminis-
tered to certain asthmatics (Baker et al., 1981; Freedman, 1977; Kochen, 1976;
Stevenson and Simon, 1981b). The first reports, generated by Kochen (1976)
and Freedman (1977), did not immediately attract much attention. However, the
simultaneous reports by Allen and Collett (198 1) and Stevenson and Simon
(1981a) at the American Academy of Allegy meetings, which linked sulfite
ingestion in foods and drugs with asthmatic episodes in several patients, sparked
considerable interest and additional research. The evidence linking ingestion of
sulfiting agents with exacerbation of asthma in a segment of the asthmatic popu-
lation is now compelling, although the role of sulfited foods in the initiation of
these reactions has not been clearly established, as will be indicated later. Addi-
tionally, sulfiting agents have been implicated in a few rare instances with other
types of hypersensitivity reactions, including anaphylactoid reactions, hypoten-
sion, and contact sensitivity (Fisher, 1975; Prenner and Stevens, 1976; Rudzki,
1979; Schwartz, 1983), indicating that asthma is not the only adverse reaction to
sulfiting agents. However, asthma is very likely to be the most common adverse
reaction to the sulfites. In this section, each of the published studies on adverse
reactions to ingestion of sulfiting agents will be reviewed, with particular empha-
sis on its contribution toward evaluating the degree of hazard posed by the use of
sulfiting agents in foods. Studies pertaining directly to respiratory exposure to
SO, will not be reviewed in detail because SO, is a well-documented hazard to
virtually all asthmatics and others when inhaled (Boushey, 1982;Koenig et al.,
1980;Linn et al., 1983;Nadel et al., 1965;Sheppard et al., 1980), and tolerance
levels have been established for exposure to SO, in the workplace and the
ambient air. However, the inhalation route of exposure may have some relevance
to the discussion because such exposures might occur from inhaling the air
released during the opening of a bag of dried fruit (Werth, 1982) or during
ingestion of an acidic beverage (Delohery et al., 1984a).
2 . The Earliest Reports
Kochen (1976)reported the case of a child with mild asthma who experienced
acute transient episodes of asthma after the consumption of sulfited foods. Con-
firmatory sulfite challenges were not conducted. This report was considered to be
an isolated, unique, and not fully substantiated case until the later reports began
to appear.
The pioneering study of the induction of asthma by ingested sulfites was
published by Freedman (1977).Freedman interviewed 272 asthmatic patients
and queried each of these patients about their asthmatic experiences following
ingestion of a particular type of orange drink. This type of orange drink, which
contains orange juice, sweetener, tartrazine, sodium benzoate, sulfur dioxide,
stabilizers, and artificial flavorings, is not available in the United States. Of the
272 patients, 30 (1 1%) reported experiencing asthma soon after ingestion of such
orange drinks. Of these 30 patients, 14 volunteered for oral challenges with
sulfur dioxide, sodium benzoate, and tartrazine. The challenges were adminis-
tered to the patients following an 8-hr period of abstinence from bronchodilators
or cromoglycate and a 3-hrperiod of abstinence from food. Sodium metabisulfite
was dissolved in a citric acid-water solution so that the challenge dose was 250
ml containing 100 ppm SO,. This would be equivalent to a dose of 25 mg of
SO,. With the addition of citric acid, the pH of the solution was acid, and
therefore most of the SO, probably existed as HSO, and H,SO,. Of the 14
patients, 8 showed a decrease in lung function as determined by a drop in their
forced expiratory volume in 1 sec (FEV,) as measured by spirometry. Any
decrease in FEV, exceeding 12% was considered positive. The group included 5
females and 3 males, and 3 of these patients also developed asthma when chal-
lenged with sodium benzoate. On challenge with SO,, the maximal drop in
FEV, occurred by 1 1 min (a range of 2-25 min) with measurable decreases often
occurring within 1-2 min. The maximal depression in FEV, ranged from 12 to
57%, with an average of 31%. Three patients had decreases in FEV, of less than
20%. One of these patients, who had a marginal drop in FEV, of 12% on
administration of 25 mg of SO,, was challenged with 75 mg of SO, and experi-
enced a decrease in FEV, of 37%. Prior administration of sodium cromoglycate
protected 4 of 4 patients from the effects of ingested SO,.
Several features of Freedman’s study are subject to criticism and possible
misinterpretation. The study is sometimes quoted as being an evaluation of the
sensitivity of 272 asthmatics to sulfiting agents. In fact, only 14 patients were
actually challenged with sulfites. Freedman used a drop of 12% in FEV, as an
indication of a positive response. This is an extremely conservative approach.
Most pulmonary specialists would consider a 12% drop as only marginal and
would require either a 15 or 20% drop to indicate a positive response. At the 20%
level, the number of responders to the 25-mg challenge would drop from 8 to 5.
Freedman did not conduct the challenges in either a placebo-controlled or dou-
ble-blind manner. Placebo control of such challenges is considered to be the
minimal safeguard against biased results and double-blind confirmation of my
reactions is preferred (Bush er al., 1986). The pH of the challenge solution may
have contributed greatly to the acquired results. SO, will be evolved from an
aqueous solution only if the pH is below 4.0. Freedman does not state the pH of
his challenge solutions. However, he prepared the solution by dissolving 0.75 g
sodium metabisulfite and 0.75 mg citric acid in 1 liter of water and then diluting
by a factor of 5. In our hands, such a solution has a pH of 2.94. At this acidic pH,
most of the free SO, would be in the HSO, form, with about 10% as H,SO,
(Green, 1976; Joslyn and Braveman, 1954). About 6% of the added meta-
bisulfite would be evolved as gaseous SO, at this pH. This would be equivalent
to 1.5 mg of SO,, a dose sufficient to induce bronchoconstrictionin asthmatics if
inhaled. Therefore, Freedman’s study may simply represent another demonstra-
tion of the ability of gaseous SO, to induce asthma.
Freedman also made some rather intriguing observations which need to be
resolved with the subsequent results of Stevenson and Simon (1981b). Freedman
observed rather rapid decreases in FEV,, with 6 of the 8 patients reaching
maximal loss of lung function within 10 min or less. By contrast, Stevenson and
Simon (1981b) measured FEV, at 30-min intervals and observed a slower re-
sponse of 15-30 min. The difference may be due to the fact that Freedman used a
beverage vehicle, while Stevenson er af. used capsules. The beverage vehicle
allowed exposure of the sublingual and buccal mucosa in addition to the gastroin-
testinal tract. The rapidity of the response suggested to Freedman that the route
of absorption of the sulfite was by inhalation of SO, vaporizing from the solution
or absorption of the sulfite through the sublingual and/or buccal mucosa. Based
on the pH of his challenge solutions, the most likely possibility is that SO, was
vaporized from these solutions and inhaled by the sensitive patients. Variable
inhalation of SO, from acidic solutions has now been demonstrated to be the
mechanism of reaction to these solutions (Delohery et a l . , 1984a,b). Another

intriguing aspect of Freedman’s work was the blockage of the response by prior
administration of sodium cromoglycate. Since cromoglycate acts by stabilizing
the mast cell membrane, thereby preventing release of histamine and other medi-
ators of the allergic response, the inhibitory action of cromoglycate would possi-
bly suggest that mediator release plays a role in the mechanism of the response to
ingested sulfiting agents. However, cromoglycate is known to have other ac-
tions, including phosphodlesterase inhibition, reduction of mucosal hyperac-
tivity, and inhibition of neurological reflexes, so blockage of histamine release
may not be the only explanation for the actions of cromoglycate.
3. 1981 American Academy of Allergy and Immunology Reports
The earliest reports by Allen and Collett (1981) and Stevenson and Simon
(1981a) were brief abstracts of presentations made at the 1981 American Acade-
my of Allergy and Immunology meeting. Allen and Collett (1981) reported 2
patients with sensitivity to sodium metabisulfite. One of these patients had had
asthmatic reactions elicited by sulfites in foods, while the other patient had
experienced asthma following administration of drugs containing sulfites. The
sensitivity to sulfites was confirmed by double-blind challenges with capsules
containing 500 mg of sodium metabisulfite. The 500-mg challenge dose is rather
high by comparison to the amounts used by Freedman (1977), Stevenson and
Simon (1981a,b), and the levels presently being used by the Australian group
(Baker and Allen, 1982; Delohery et a l . , 1984a,b). The patients described here
were also sensitive to tartrazine, aspirin, and sodium benzoate. Stevenson and
Simon (1981a) identified 4 asthmatic patients with sulfite sensitivity. They also
employed capsule challenges, but used potassium metabisulfite. The threshold
doses for decreases in lung function ranged from 10 to 50 mg. These patients
were not found to be sensitive to sodium benzoate, aspirin, tartrazine, or mono-
sodium glutamate.
4 . Further Reports from Australia
A later report by Allen’s group describes in detail the cases of 2 sulfite-

sensitive patients (Baker et a l . , 1981). It is not clear if these patients are identical
to the ones described in the earlier abstract. The first case was a 67-year-old
female who had experienced asthma after ingesting a crabmeat salad prepared
with vinegar dressing. A subsequent challenge of this patient with capsules of
sodium metabisulfite confirmed the existence of an asthmatic reaction related to
the consumption of sulfites. The second case was a 23-year-old female whose
asthmatic symptoms worsened on ingestion of wine. A subsequent challenge
with a capsule containing 500 mg of sodium metabisulfite confirmed the exis-

tence of the asthmatic reaction to sulfites. The challenges were done double
blind, with lactose as the negative control.
A third report from the Australian group, also in abstract form, details their
experiences with metabisulfite challenges through early 1982 (Baker and Allen,
1982). By this time, they had identified 8 patients with asthmatic sensitivity to
oral challenge with metabisulfite (presumably the sodium salt). Of the 8 patients,
3 were also sensitive to aspirin and other food additives, including tartrazine and
benzoate. The challenge protocol had been modified to include administration of
graded doses starting at 10 mg and progressing through 300 mg, with lung
function evaluations at 0.5-hr intervals. Curiously, 4 of the 8 sensitive patients
did not react to a 300-mg capsule challenge, but did react to a 25-mg challenge of
metabisulfite dissolved in 50 ml of 0.5% citric acid. This mode of administration
is quite similar to that of Freedman (1977). The reactions to acidic sulfite solu-
tions occurred within 1-5 min, while positive capsule challenges showed a 20- to
30-min lag period. They conclude that the response to acidic sulfite solutions is
due to inhalation of vaporized SO,.
More recent reports from the Australian group (Delohery et af., 1984a,b)
delve more deeply into the comparative responses to capsule versus beverage
challenges. Acidic solutions of metabisulfite were able to provoke asthma in
60% of all asthmatics, a much higher percentage than found with capsule chal-
lenges (Delohery et al., 1984a). A comparison of sulfite reactors with asthmatics
not reactive to sulfites revealed that both groups were equally sensitive to inhaled
SO,, but that the sulfite reactors were the only group responsive to ingestion of
sulfited acidic beverages. The sulfite reactors responded to a mouthwash with a
sulfite solution, but not to sulfite solution administered directly into the stomach
via a nasogastric tube. It must be assumed that this group of reactive asthmatics
does not have any capsule reactors because they would be predicted to respond to
any direct gastric challenge. Delohery et al. (1984a.b) conclude that the bev-
erage reactors are inhaling SO, as they swallow, while nonreactors can swallow
without inhalation. Allen and Delohery (1985) revealed that these asthmatics do
not respond to sulfited acidic beverages if they take a deep breath and hold it
before using a sulfite mouthwash. The existence of such a high percentage of
beverage reactors is somewhat surprising, although all asthmatics respond to
inhaled SO,. However, the practical significance of this type of sulfite sensitivity
is uncertain. Delohery et af. (1984a,b) used challenges of 50 mg of metabisulfite
in a citric acid solution. It is unlikely that asthmatics would routinely encounter
such levels of free sulfite in most beverages. Wine might easily contain 50 mg of
total sulfite per serving, but the majority of this sulfite would be in the form of
combined sulfites. Still, this type of sensitivity may explain the common com-
plaints of asthmatics about adverse reactions to the ingestion of wines.
Allen and Delohery ( 1985) also investigated the mechanism involved in reac-
tions to sulfite in capsules. After ingestion of 25- to 50-mg capsules of meta-
bisulfite, 4-50 ppm of SO, could be detected in the stomach via a nasogastric
tube. They speculate that SO, is evolved from metabisulfite by the action of
stomach acid and that the SO, can be inhaled following eructation. Unfortunate-
ly, they did not measure SO, concentrations in the nasopharynx after capsule
ingestion.
5 . Further Reports from the La Jolla Group
Stevenson and Simon (1981b) also published a more detailed account of their
initial findings. Descriptions of 5 sulfite-sensitive patients are provided in this
report. Four of the patients were identical to the ones described in their earlier
abstract. The challenges were performed with capsules of potassium meta-
bisulfite. Graded doses starting at l mg were employed, with the doses increas-
ing to 5, 10, 25, and 50 mg of K,S,O, until an asthmatic response was noted.
The 5 patients had asthmatic reactions beginning at 15-30 min after administra-
tion of the threshold dose. The threshold dose was 10 mg for 2 of the patients, 25
mg for another 2 patients, and 50 mg for the fifth patient. Since several doses
were administered at 30-min intervals, it is possible, though unlikely, that the
patients were reacting to an accumulated dose rather than the last dose adminis-
tered. Falls in FEV, ranged from 23 to 49%. The challenges were placebo
controlled, reproducible, and blinded to some.extent. This experimental design
was imperative, since all of these patients were severe asthmatics who required
steroids for control. Such asthmatics would be predicted to be unstable, so repeat
challenges and blinded challenges were necessary. These patients were not sen-
sitive to aspirin, tartrazine, or monosodium glutamate.
Stevenson and Simon (1981b) attempted unsuccessfully to define the mecha-
nism of action of potassium metabisulfite in these patients. Evidence for an IgE-
mediated reaction could not be found. In fact, no evidence could be found that
mediator release is involved in the reaction. Cutaneous testing with 0.02 mg of
K,S,O, given intradermally was negative in the 4 tested patients. Incubation of
peripheral basophils with K,S,O, in concentrations up to 0.01 M failed to induce
histamine release. These tests would be positive in reactions involving mediator
release whether IgE-mediated or not. Despite the lack of evidence for an IgE-
mediated reaction among the patients studied by Stevenson and Simon (1981b),
systemic sensitivity beyond altered lung function was noted in all of their pa-
tients. The systemic symptoms were flushing, weakness, and hypotension.
These symptoms can be involved in IgE-mediated reactions or other reactions
involving mediator release. Stevenson and Simon (1981b) hypothesize that po-
tassium metabisulfite acts via stimulation of the cholinergic reflex arc. This
stimulation would account for some of the observed symptoms, including bron-
choconstriction. However, it is difficult to explain hypotension on this basis. The
therapeutic effectiveness of atropine is also consistent with this mechanism.
Some evidence suggests that inhaled SO, activates irritant receptors in the bron-
chial tubes and that these receptors may activate the cholinergic reflex arc
(Boushey, 1982; Nadel et al., 1965). However, other theories of the actions of
inhaled SO, also exist (Boushey, 1982). Further proof will be needed before
cholinergic stimulation will be accepted as the mode of action of ingested
sulfites.
In a 1981 abstract from the American Academy of Allergy and Immunology
meeting, Simon et al. (1982) presented the first indication of the prevalence of
sensitivity to ingested sulfites among asthmatics. A total of 61 asthmatics chosen
randomly were challenged with potassium metabisulfite capsules containing 10,
25, 50, 100, and 200 mg K,S,O, at 30-min intervals. A positive reaction was
defined as a fall in FEV, of at least 25%. Challenges were placebo controlled and
single blind, with repetition of any positive response in a second challenge. Of
the 61 patients, 5 (8.2%) reacted to K,S,O,. The reactions were milder than
those encountered in their earlier studies (Stevenson and Simon, 1981a,b), and
the threshold doses tended to be higher. This study would suggest that the
prevalence of sulfite sensitivity among asthmatics is rather high. However, we
question whether the population of asthmatics used in this survey was truly
random. Many of the asthmatics used in this survey had severe asthma, and the
study group was probably not a true cross section of the entire asthmatic popula-
tion.
The La Jolla group presented three abstracts at the 1984 American Academy of
Allergy and Immunology meeting (Goldfarb and Simon, 1984; Jacobsen et al.,
1984; Simon et al., 1984). Goldfarb and Simon (1984) evaluated the com-
parative sensitivities of sulfite-sensitive asthmatics (SSA) as a function of the
route of exposure. Six SSA were used in this study; all 6 SSA had reacted to
capsule challenges with 10-50 mg of sulfite, with a fall in FEV, of >25%. The
minimum provoking dose for a beverage challenge was approximately one-half
that of the capsule challenge. Inhalation of nebulized sulfite solutions provoked
reactions at one-tenth to one-one hundredth of the capsule challenge dose. None
of these SSA reacted to subcutaneous administration of sulfites at doses up to 10
times higher than their provoking capsule dose. Obviously, inhalant exposures
are the most hazardous to SSA. Inhalant exposures could be encountered through
the use of bronchodilator solutions preserved with sulfites (Koepke et al., 1983).
Usually, the bronchodilating effect of the active ingredient would overwhelm the
bronchoconstricting effect of sulfite, although a few patients seem to suffer
paradoxical bronchoconstriction when treated with sulfited bronchodilators
(Koepke et al., 1984a; Simon, 1985).
Jacobsen et af.(1984) focused their efforts on elucidation of the mechanism of

sulfite sensitivity in asthmatics. Using skin biopsies from SSA, Jacobsen et al.
( 1984) cultured skin fibroblasts and demonstrated that these cells had diminished
levels of sulfite oxidase by comparison to cells cultured from normal individuals
and asthmatics without sulfite sensitivity. The depressed levels of sulfite oxidase
may indicate that these individuals are heterozygous for a deficiency of the
enzyme. The diminished sulfite oxidase levels could compromise the detoxifica-
tion of sulfite in these individuals (see Section IILA), but further studies will be
needed to define the full implications of this finding.
Jacobsen et af. (1984) were also able to show that cyanocobalamin (vitamin
B,,) can protect SSA from the effects of ingested sulfite. Up to 50 mg of vitamin
B,, orally was necessary to block the reaction to ingestion of a 50-mg capsule of
K,S,O,. The vitamin B,, action was catalytic, as demonstrated by the observa-
tion that 5 mg would protect against 50 mg of K,S,O,. The B,, effect is
probably associated with the known ability of the cobalamins to catalyze the
oxidation of sulfite to sulfate (see Section II,F,6). The effective dose of vitamin
B,, is far in excess of the recommended dietary allowance for this vitamin. It is
even in excess of the levels of vitamin B,, used to treat pernicious anemia.
However, such pharmacological doses of vitamin B,, may provide a convenient
means of prophylaxis for SSA. R. A. Simon (personal communication) is now
counseling his SSA patients to take 5 mg of vitamin B,, before eating a restau-
rant meal.
Simon et al. (1984) evaluated the effectiveness of a variety of possible block-
ing agents on sulfite-induced asthma among SSA. Vitamin B,,, atropine, cro-
molyn, and doxepin were all effective blocking agents. These agents were effec-
tive irrespective of the route of administration of the sulfite. The effectiveness of
all four agents is rather surprising, since they have different modes of action.
Atropine is an anticholinergic agent, cromolyn is a mast cell membrane stabilizer
and calcium channel blocker, doxepin is a broad spectrum antihistamine, and
vitamin B,, catalyzes sulfite oxidation. Rather high doses of these blocking
agents were necessary, and it is possible that at such high doses these agents
could have additional effects beyond those just mentioned. This experiment does
not provide many clues to the mechanism of action of sulfites in provoking
asthma in these subjects. The equivalent effectiveness of these agents toward
ingested versus inhaled sulfite is also rather surprising, since the mechanisms of
the two routes of exposure are almost certain to be different.
At the 1985 American Academy of Allergy and Immunology meeting, this
group presented two additional abstracts on sulfite sensitivity (Howland and
Simon, 1985; Simon, 1985). One of these reports involved the description of two
cases of paradoxical bronchoconstriction in sulfite-sensitive asthmatics after ad-
ministration of a sulfited bronchodilator (Simon, 1985). Howland and Simon
(1985) challenged 5 sulfite-sensitive asthmatics with 3 oz of sulfited lettuce

containing 80-90 mg of bisulfite (calculated by assessing the amount of sulfite
solution not recovered after drainage). All 5 patients experienced pronounced
decreases in lung function; the mean FEV, decrease was 44%, with a range of
31-64%. Untreated lettuce had no effect. This challenge study demonstrates
conclusively that sulfited lettuce can elicit asthmatic reactions. Whether other
sulfited foods will elicit these reactions is not known. Sulfited lettuce contains
appreciable quantities of free SO, (Taylor et al., 1985), which may enhance the
likelihood that lettuce will initiate asthmatic reactions by comparison to other
sulfited foods.
6. Other Reports of Sulfite-Induced Asthma
Several additional studies on the prevalence of sulfite sensitivity were con-

ducted after Simon et al. (1982) reported that 8.2% of all asthmatics might be
affected. Bush et al. (1985) showed that sensitivity to encapsulated sulfites was
an appreciable risk only for those patients who require steroids for the control of
their symptoms. Among 83 steroid-dependent or severe asthmatics, the preva-
lence of sulfite sensitivity was 8.4%. Among 120 mild or nonsteroid-dependent
asthmatics, the prevalence of sulfite sensitivity was only 0.8%. Mild asthmatics
make up about 80% of all asthmatics, so the overall prevalence for the total
population of asthmatics is estimated to be 1.8% from this study. Buckley et al.
(1985) selected 134 patients from a total clinic population of 1073 asthmatic
subjects; 50/134 or 37% reacted to oral challenges with capsules of K,S,O,.
This suggests a minimal prevalence of 4.6% (50/1073). However, as with the
population examined by Simon et al. (1982), there is no indication that the
patient population evaluated by Buckley et al. (1985) is representative of the
overall asthmatic population. Towns and Mellis (1984) performed oral sulfite
challenges with both capsules and citric acid solutions of Na,S,O,. None of the
children developed asthma after challenge with capsules, but 19 of 29 (66%)
experienced a significant decrease in FEV, after challenge with an acidic sulfite
solution. This confirms previous suggestions that many more asthmatics are
sensitive to acidic solutions of sulfite by comparison to encapsulated sulfites
(Delohery et al., 1984a,b).
Other case reports of asthmatic sensitivity to sulfites have also appeared
(Sprenger et al., 1985; Altman et al., 1985; Schwartz and Chester, 1984;
Koepke et al., 1984; Yang et al., 1985; Werth, 1982; Twarog and Leung, 1982).
One was a patient with a history of asthma that worsened with ingestion of
certain foods, particularly dried apricots and Catawba grape juice (Werth, 1982).
The patient also experienced flushing during these episodes. Both of these foods
are sulfited. Occasional asthmatic attacks were experienced following ingestion
of wine, beer, cheese, blueberries, apples, and strawbenies. Of these foods, only
wine, beer, and possibly freshly cut fruits would be expected to contain residual
SO,. Symptoms of asthma were produced in the patient by sniffing a freshly
opened bag of dried apricots. Oral challenge with capsules of potassium meta-
bisulfite at doses up to 50 mg were negative. Inhalation of nebulized K,S,05 in
water induced a rapid decline in FEV, . Apparently, this patient is another exam-
ple of an individual who responds to inhaled SO, but not to ingested sulfites. He
constitutes further proof for our suggestion that two groups of sulfite-sensitive
asthmatics exist.
Another case was reported by Twarog and h u n g (1982). This patient had
perennial asthma and had experienced several adverse reactions to drugs that
contained sodium bisulfite or sodium metabisulfite. Oral challenge of this patient
with sodium metabisulfite in water revealed that a 5-mg dose caused a 52% drop
in FEV,. The reaction to a 5-mg dose makes this patient the most sensitive
described so far. Flushing was also noted. In addition, this patient may be
unique, since evidence of mediator release in response to the sulfites was ob-
tained in her case. Skin testing with sodium bisulfite at 0.1 mg/ml resulted in a
definite wheal and flare reaction. Sodium bisulfite at concentrations of lop3-
10- M also caused release of histamine from this patient’s leukocytes. For both
skin testing and leukocyte histamine release, control tests on other individuals
were negative. These findings do not constitute proof for the existence of an IgE-
mediated or type I reaction, however, because no evidence for the existence of a
specific antibody was obtained. However, this patient seems to be unique, since
Stevenson and Simon (198 lb) found no evidence of mediator release in 4 of their
sulfite-sensitive patients. This patient probably represents a small subgroup of
sulfite-sensitive patients. Apparently, the majority do not react via mediator
release, but obviously some patients may mount such responses. This patient was
challenged with sodium metabisulfite in water, a slightly acidic solution. It is
difficult to determine if her response was due to inhalation of SO, or ingestion of
sulfites. The ingestion route would seem most probable, since a 10-min lapse
occurred between administration of the dose and the fall in FEV,. Also, SO,
would not be evolved from a water solution, which would have a pH of greater
than 4.0.
Altman et al. (1985) and Sprenger et al. (1985) provide some additional
evidence for the possibility of mediator release in the pathogenesis of sulfite-
induced asthma. Sprenger et al. (1985) describe a single patient with sensitivity
to both inhaled SO, and aqueous solutions of K,S,O, (the pH was not specified).
In this patient, an increase in the level of neutrophil chemotactic activity (NCA)
in the serum was observed 2 hr after the maximal decline in FEV, . Altman et al.
(1985) identified 3 additional patients with similar patterns of sensitivity along
with increased serum NCA. NCA can be released from mast cells with appropri-
ate antigen challenges. However, these findings are somewhat confusing. The
increase in NCA in serum did not correspond in time to the decreased lung
function. Also, the pH of the sulfite solutions is not provided, so it is impossible
to know if these patients fall in the small group with sensitivities to encapsulated
sulfites or the large group with sensitivities to ingestion of acidic sulfited
beverages.
The data from Sprenger et al. (1985) suggest that patients with sensitivities to
ingested sulfites would also display inhaled sulfite sensitivity. Koepke et al.
(1984b) performed inhalation challenges on 3 sulfite-sensitive (by capsule chal-
lenge) and 10 nonsulfite-sensitive asthmatics. All 3 sulfite-sensitive asthmatics
and 4 of the 10 others had declines in FEV, of 20% or greater. The remaining 6
asthmatics had diminished lung function also, but it had not reached a 20%
decrease at the administered levels of sulfite. Again, these data suggest that all
patients with reactions to ingested sulfites will respond to inhaled sulfites.
Schwartz and Chester (1984) obtained some conflicting information. Six asth-
matics who developed airway obstruction after ingesting solutions of K,S,O,
were subjected to inhalation challenge. Only 3 of the 6 patients responded to both
ingestion and inhalation challenges with sulfite. These data suggest that a
positive oral sulfite challenge is usually but not invariably accompanied by a
positive aerosol challenge.
Yang et al. (1985) identified 3 sulfite-sensitive asthmatics using oral chal-
lenges with K,S,O, capsules. Two of the patients had positive intradermal skin
tests to 1 mg/ml solutions of K,S,O,. Passive transfer was also demonstrated
with unheated serum from one of these patients. They conclude that IgE mecha-
nisms may play a role in a subset of sulfite-sensitive asthmatics.
Several reviews on asthmatic reactions to sulfites have appeared (Bush et al.,
1986; Schwartz, 1984; Simon, 1984; Stevenson and Simon, 1984; Twarog,
1983).
7. Other Adverse Reactions to Sulfites
Asthma has not been the only adverse reaction associated with ingestion of
sulfites, although it appears to be the most common. Prenner and Stevens (1976)
reported a patient who experienced urticaria and pruritis, swelling of the tongue,
difficulty in swallowing, and tightness in the chest after ingestion of a sulfited
restaurant salad. The patient had a positive scratch test to 0.2 mg of sodium
bisulfite. An oral challenge with 10 mg of sodium bisulfite produced itching,
nausea, flushing, cough, tightness in the throat, and erythema. Passive transfer
testing was also positive. The passive transfer test indicates the presence of a
serum factor involved in this patient’s response to sulfites. However, even in this
case, this cannot be construed as definite evidence of an IgE-mediated reaction,
although it is suggestive of such a reaction. Prenner and Stevens (1976) men-

tioned in their report that several food handlers had described instances of contact
sensitivity from handling sulfite solutions. Fisher (1975) had previously reported
a case of eczema in a food handler, which had been attributed to bisulfite
exposure. Rudzki (1979) recently identified sulfites as contact allergens as well.
Several other cases of urticaria and angioedema attributed to sulfites have been
described (Allen et al., 1984; Habernicht et al., 1983; Huang and Fraser, 1984).
Habernicht et al. (1983) described two women who reported urticaria and an-
gioedema after ingestion of sulfited foods. One of these patients developed
urticaria and burning of the scalp within 15 min following challenge with 25 mg
K,S,O, in a capsule. Allen et al. (1984) note that urticaria can be induced by
sulfite challenges, but that larger doses are usually required than those used in
challenges of asthmatic subjects. Huang and Fraser (1984) suggested that sub-
cutaneous administration of sulfites could provoke urticaria, angioedema, and
laryngeal edema in sensitive individuals. Subcutaneous injection of 1.8 ml of
lidocaine, which contains 0.5 mg of NaHSO,, produced palmar pruritis in a
patient. No controlled challenge was administered. Yang et al. (1985) described
a single patient with urticaria and angioedema after oral challenge with K,S,O,
capsules.
Very recently, another type of adverse reaction to sulfites has been described
(Schwartz, 1983). Two patients were identified with anaphylactic-like reactions
possibly associated with restaurant meals. The first patient had experienced an
episode of clammy skin, weakness, headache, chest tightness, tachycardia, and a
feeling of dissociation from his body commencing 10 min after eating a restau-
rant salad. The second patient had developed dizziness, nausea, palpitations,
hives, dysphagia, chest tightness, and dyspnea after a restaurant meal of shellfish
and salad. Both patients were administered single-blind, placebo-controlledchal-
lenges with metabisulfite (Na or K salt not specified). With increasing doses in
the range of 10-50 mg, progressively worsening hypotension was observed in
both patients. Abdominal distress, nausea, dizziness, and weakness were also
noted. Not all of the symptoms from the restaurant episodes were seen in the
challenges, but this may have been due to the exposure to lower doses of sulfites
in the challenges. Neither patient experienced asthma and neither had positive
skin tests, so these reactions were not IgE mediated. These cases are the first
reports of hypotension without asthma following challenge with sulfites. Steven-
son and Simon (1981b) noted hypotension in some of their patients who also
experienced asthma on challenge with sulfites. The frequency of the hypotensive
response to sulfites is unknown.
Sulfiting agents may also cause problems when administered as a component
of a drug formulation. The most common manifestation is asthma, as described
previously. The use of bisulfite in epidural anesthetics has recently been associ-
SULLlTES IN FOODS 59
ated with paralysis of the lower extremities (Wang et al., 1984). This rare
reaction occurs when the anesthetic is accidentally injected into the subarachnoid
space. The paralytic condition was duplicated in rabbits by injecting 1.2-2.4 mg
of sodium bisulfite into the lumbar subarachnoid space.
Flaherty et al. (1985) described an unusual case of sulfite sensitivity in a
patient with underlying liver disease (sclerosing cholangitis) and ulcerative col-
itis. This patient’s liver condition was observed to worsen after ingestion of
home-preserved juices and restaurant salads. These episodes were often accom-
panied by palmer and plantar erythema with pruritis. The liver function tests in
this patient improved on a sulfite-free diet. An increase in serum levels of liver
enzymes was noted after challenge with 500 mg of metabisulfite. This increase
could be blocked by prior administration of 3 mg of vitamin B,*.
Sulfites have been evaluated for their possible role in other conditions as well.
Sonin and Patterson (1985) failed to trigger episodes of idiopathic anaphylaxis in
12 patients using oral challenges with Na,S,O, in lemonade. Similarly, Meggs et
al. (1985) could find no role for sulfites in the elicitation of idiopathic anaphylaxis
in challenges of 25 patients with capsules of NaHSO,. However, plasma his-
tamine levels were elevated twofold in 23 of the 25 patients following bisulfite
challenge. Eight patients with systemic mastocytosis were subjected to similar
challenges, and no evidence was found to implicate sulfites in this condition
(Meggs et al., 1985). Like the patients with idiopathic anaphylaxis, plasma
histamine levels were elevated twofold in 7 of the 8 patients with systemic
mastocytosis after bisulfite challenge.
8. Sensitivity to Suljited Foods
Many of the reported sulfite-sensitive asthmatics provide a history of asth-

matic reactions to foods that are suspected to contain sulfite residues. Their
sensitivities to free inorganic sulfites have been documented through capsule
and/or beverage challenges. However, there are only two reports of controlled
challenges to a sulfited food or beverage unless one wants to count the challenges
performed with sulfites in citric acid solutions or lemonade. We do not believe
that the challenges with citric acid solutions are representative of the situation
that exists with most foods, since most sulfited foods have pHs above 4.0 and
would not spontaneously liberate SO,. The first study of the sensitivity of asth-
matics to sulfited foods or beverages was conducted by Seyal et al. (1984) with
wine. The subjects were asked to drink 4 oz of white wine containing 140
mg/liter of SO,. Only 1/25 asthmatics and 0/25 controls developed asthma
following the challenge. Unfortunately, Seyal et al. (1984) did not prescreen
their asthmatic population for sulfite senstivity, so the number of sulfite-sensitive
asthmatics in their group is unknown and probably small. The study would have
been strengthened considerably if it had been conducted on a group of sulfite-

sensitive asthmatics. Also, Seyal et al. (1984) considered a drop in FEV,of 12%
or greater as a positive response. As noted previously, most pulmonary spe-
cialists would require a drop of at least 15-20% to signal a positive response.
Therefore, the single responder in this study may be questionable. The other
challenge study of sulfited food was conducted by Howland and Simon (1985)
with sulfited lettuce; the results were described earlier.
As discussed earlier, SO, and the inorganic sulfites react rapidly with food
components. The rate, completeness, and products formed by these reactions are
dependent on the pH, temperature, sulfite concentrations, type and concentration
of various food components, and other factors. The primary products in many
foods are the hydroxysulfonates of aldehydes, ketones, and reducing sugars. In
vitro experiments have shown that these sulfite addition compounds are rather
stable in dilute acid at room temperature (Adachi et al., 1979; Burroughs and
Sparks, 1973; Green, 1976; Joslyn and Braverman, 1954). Therefore, they
would not be expected to liberate SO, in the stomach under its acidic conditions.
Some release of sulfites from sugar hydroxysulfonates might be predicted to
occur in the neutral pH conditions of the small intestine. However, other hy-
droxysulfonates would be stable even under these conditions. Very recent work
indicates that one hydroxysulfonate is not metabolized at all in rats or mice after
feeding in the diet (Walker et af., 1983a). Sulfite addition compounds can also
be formed with amino acids and proteins (Green, 1976; Schroeter, 1966). The
stability of these adducts in gastric acid is not known, but they are probably more
stable than many of the hydroxysulfonates. The question then centers on the role
of the sulfite addition products in the induction of the asthmatic response. The
answer to that question is not known.
Some added SO, and inorganic sulfites remain in the food product in the
uncombined state. This free SO, would probably react much like the sulfites
ingested in capsules. Only if the food was below pH 4.0 would the gas, SO,, be
evolved from the food in the oral cavity. In other foods, free SO, would exist
primarily as HSO, and SO:-. Gaseous SO,, if it exists in the food, would
likely pose a hazard to asthmatics who might inhale it during consumption of the
food (Delohery et al., 1984a,b; Towns and Mellis, 1984). Other forms of free
SO, would pose a possible hazard only to those individuals with sensitivites to
sulfites in capsules. The combination of sulfites with food components would
drastically lower the free SO, content of most sulfited foods (lettuce is an
exception), thereby limiting exposure to these free forms of the sulfiting agents.
Even with the free sulfites, the food matrix may diminish the degree of sen-
sitivity by slowing rates of absorption and access to the sites of action.
The degree of hazard posed to sulfite-sensitive asthmatics by sulfited foods
can only be established with actual food challenges of sensitive patients. Since
foods vary in the nature of their combined sulfites and in the amount of residual
free sulfites, such challenges will need to be performed with a variety of sulfited
foods. We expect, on the basis of chemical considerations, that most sulfited
foods will be much less hazardous than equivalent amounts of sulfites in cap-
sules. Again, sulfited lettuce may be an exception, since it contains a high
proportion of free SO, (Taylor et al., 1985).
IV. POSSIBLE SUBSTITUTES AND THEIR LIMITATIONS
If the current GRAS review leads to some limitation on the continued use of
sulfites, it will be necessary to consider alternatives. This section is designed to
present some possible alternatives and their limitations. A complete substitute for
the sulfiting agents which would possess all of the desirable properties of this
group of food additives will be virtually impossible to find. Replacements for
each of the individual benefits provided by the sulfiting agents might be identi-
fied. However, in many foods, sulfiting agents are used for more than one
purpose, e.g., the use in white wines for both its antimicrobial and antibrowning
properties. The potential substitutes are also less effective and more costly in
most cases. Many of the suggestions presented in this section were obtained from
the review by Roberts and McWeeny (1972).
A. CONTROL OF ENZYMATIC BROWNING
Enzymatic browning will be inhibited by any process that destroys or inacti-

vates the enzyme. Blanching would obviously work, but is impractical for use on
fresh fruits and vegetables. Acid denaturation of the enzyme is also feasible,
e.g., with application of lemon juice or vinegar. Since polyphenoloxidase is
dependent on cupric ions for activity, the removal of these metallic ions may
inhibit the process. EDTA would serve this purpose. Citric acid and tartaric acid
also work in this manner. The activity of the enzyme can be slowed by lowering
the pH through the addition of acids or fermentation. Lowering the water activity
of the food can also diminish this reaction, but again dehydration is often not
practical. Removal of oxygen works quite well, since oxygen is a required
substrate for polyphenoloxidase. Reducing agents can be effective in converting
the quinones back to the diphenols. Ascorbate and cysteine have been used in
this manner. On cut surfaces, the sulfites have the advantage of being able to
penetrate quickly into the cellular matrix, a property not shared by their sub-
stitutes. Hence, the substitutes are less effective.
Several alternatives using combinations of the above materials for control of
enzymatic browning have been developed. One procedure involves ascorbate
62 STEVE L . TAYLOR ET AL.
and CaCl,. Another alternative is a combination of phosphate, citric acid, dex-

trose, aluminum sulfate, and sodium erythrobate. Both of these alternative meth-
ods work primarily on the basis of acidification with reducing activity. Ponting er
al. (1971) pioneered use of ascorbate and calcium in the preservation of sliced
apples. Montgomery (1983) recently noted that enzymatic browning of pear juice
concentrate can be prevented by cysteine. Cysteine does not work well on cut
surfaces because of its lack of cellular penetration.
B. CONTROL OF NONENZYMATIC BROWNING
Nonenzymatic browning can be controlled by (1) elimination of the active

compounds, (2) lowering pH, (3) separation of the active species, or (4) behydra-
tion to low water activities. The removal of sugars can be be effected by fermen-
tation, glucose oxidase, or leaching, as is done with potatoes. Theoretically,
cysteine could be effective in competing for reaction with reducing sugars, but
this has never been evaluated as a practical alternative. The browning reaction
can also be slowed by the addition of acids such as lactic, tartaric, citric, acetic,
or ascorbic acids, or aluminum sulfate. Physical separation of the active species
can occasionally work if, e.g., the sugar is in the sauce and the amino acid is in
the entree. Dehydration to less than 4% water will also inhibit nonenzymatic
browning, but is not economically feasible. The replacement of sulfites in the
control of nonenzymatic browning will be difficult because none of the above
treatments is as effective or universally applicable.
C. USE AS ANTIOXIDANTS OR REDUCING AGENTS
Ascorbic acid can replace sulfites as antioxidants in beer, but naturally occur-
ring levels of SO, in beer may make replacement unnecessary. Sulfites have the
added advantage of controlling nitrosamine formation in the malt (Lukes et al.,
1980). As a reducing agent, it may be possible to replace sulfites with cysteine or
other mercaptans, although these substitutes have undesirable organoleptic prop-
erties and undesirable color and texture.
D. USE AS AN ANTIMICROBIAL AGENT
For wines and corn steep liquors, alternative agents will be difficult to find.
Other antimicrobial agents will inhibit undesirable fermentations, but all have
drawbacks in terms of expense, stability, specificity, or objectionable off-fla-
vors. Agents such as lactic acid or sorbic acid may be useful in lowering the
necessary levels of SO,. In table grapes, the gaseous nature of SO, is indispens-
able and a substitute will be difficult to identify.
E. USE AS A BLEACHING AGENT
For the bleaching of cherries in the production of maraschino cherries, SO,

has no equal. Sodium chlorite is useful for secondary bleaching (Beavers and
Payne, 1968), but is far inferior for primary bleaching.
V. FUTURE RESEARCH NEEDS
Despite their long history of use as food additives, much remains to be learned
about sulfites which would be helpful to the present concerns about their safety.
Better information is needed on the issues of consumer exposure assessment and
the toxicity and hypersensitivity reactions to sulfited foods.
For the purpose of improving consumer exposure assessments, better analyt-
ical methods and more analytical data are needed. The methods should empha-
size determination of both free and combined sulfites. In particular, better meth-
ods are needed for the determination of combined or total sulfites. The combined
sulfites appear to be less toxic than the free sulfites, so analytical data for both
free and combined (or total) sulfite are needed. The analytical data should em-
phasize samples taken from typical points of consumption so that the losses on
storage and preparation can be taken into account. As part of this effort, further
investigations into the fate of sulfites in specific foods are needed. Emphasis
should be placed on the identification of combined forms of sulfite so that their
toxicity might be evaluated.
From the viewpoint of toxicity assessment, further work is especially needed
on the assessment of the toxicity of the combined sulfites. Since the bulk of
sulfite ingestion is in the form of combined sulfites, the general lack of such
information makes hazard evaluation virtually impossible. The toxicological
studies should probably be focused on chronic and subchronic toxicity and
should emphasize the oral route of administration. Further toxicological com-
parisons are needed in sulfite oxidase-deficient versus normal animals.
On the hypersensitivity issue, a variety of unknowns remain. The major issue
will be the determination of the responsiveness of sulfite-sensitive asthmatics to
sulfited foods in controlled challenge trials. Only through the use of such chal-
lenges will the tolerance of these asthmatics for sulfited foods become available.
In all likelihood, many sulfited foods will contain such low residual levels that
they will not elicit asthma in these patients. The issue of the incidence of sulfite
sensitivity in the asthmatic population remains to be answered as well. The
incidence among mild asthmatics is unknown, although it appears as though the
severe asthmatics are most likely to be sulfite reactors. The existence of more
than one type of sulfite-sensitive asthmatic, acidic beverage reactors versus
capsule reactors, seems likely from present data, but more studies are needed to
establish the mechanisms (e.g., site of exposure) responsible for the existence of
these two groups. The mechanism of action of encapsulated sulfites in inducing
asthma in some asthmatics remains a mystery. Effective treatment may depend
on the elucidation of this mechanism. Lastly, the existence of other types of
hypersensitivity responses to sulfites has been established, but more studies are
needed to establish the prevalence of such reactions.
REFERENCES
Adachi, T., Nonogi, H., Fuke, T., Ikuzawa, M., Fujita, K., Izumi, T., Hamano, T., Mitsuhashi,
Y.,Matsuki, Y.,Suzuki, H., Toyoda, M., Ito, Y.,and Iwaida, M. 1979. On the combination
of sulphite with food ingredients (aldehydes, ketones, and sugars). Z. Lebensm. Unters. Forsch.
168, 200-205.
Allen, D. H., and Collett, P. 1981. Life-threatening asthmatic reactions to the food and drug
preservative, sodium metabisulfite. J . Allergy Clin. Immunol. 67, 7 I .
Allen, D., and Delohery, J. 1985. Metabisulfite induced asthma. J . Allergy Clin.Immunol. 75, 145.
Allen, D. H., Van Nunen, S., Loblay, R., Clarke, L., and Swain, A. 1984. Adverse reactions to
foods. Med. J. Ausr. 141, S 3 7 4 4 2 .
Altman, L. C., Sprenger, J. D., Marshall, S., Johnson, L. E., Koenig, J., and Pierson, W. E. 1985.
Neutrophil chemotactic activity in sulfite sensitive patients. J . Allergy Clin. Immunol. 75, 144.
American Conference of Governmental Industrial Hygients. 1982. Threshold limit values for chem-
ical substances in work air. Am. Conf. Govt. I d . Hyg., Cincinnati. Ohio.
Anderson, C., Warner, C. R.,Daniels, D. H., and Padgett, K. L. 1983. Analysis of sulfites in foods
by ion chromatography. AOAC Annu. Meet., 97th. Washington, D . C . , Ocr. 6.
Anonymous 1984. BATF proposes lowering sulfite content of wine. Food Chem. News Oct. 1, 20-
22.
Applebaum, R. S., and Marth, E. H. 1982. Use of sulphite or bentonite to eliminate aflatoxin MI
from naturally contaminated raw whole milk. Z . Lebensm. Unters. Forsch. 174, 303-305.
Baker, G. J . , and Allen, D. H. 1982. The spectrum of metabisulfite induced asthmatic reactions;
their diagnosis and management. Ausr. N. Z. Med. J . 12, 213.
Baker, G. J., Collett, P., and Allen, D. H. 1981. Bronchospasm induced by metabisulfite-containing
foods and drugs. Med. J. Aust. 2, 614-616.
Baloch, A. K . , Buckle, K. A., and Edwards, R. A. 1977. Stability of @-carotenein model systems
containing sulphite. J. Food Technol. 12, 309-316.
Beavers, D. V., and Payne, C. H. 1968. Upgrades brined cherries. Food Eng. 40, 84-85.
Beems, R. B., Spit, B. J., K&ter, H. B. W. M., and Feron, V. J. 1983. Nature and histogenesis of
sulfite-induced gastric lesions in rats. Exp. Mol. Parhol. 36, 3 16-325.
Beutler, H. 0. 1984. A new enzymatic method for determination of sulphite in food. Food Chem.
15, 157-164.
Bhagat, B., and Luckett, M. F. 1960. The absorption and elimination of metabisulphite and thiosul-
fate by rats. J . Pharm. Pharmacol. 12, 690-694.
Bhagat, B., and Lockett, M. F. 1964. The effect of sulphite in solid diets on the growth of rats. Food
Cosmet. Toxicol. 2, 1-13.
Boushey, H. A. 1982. Bronchial hyperreactivity to sulfur dioxide: Physiologic and political implica-
tions. J. Allergy Clin. Immunol. 69, 335-338.
Braverman, B., Shapiro, R., and Szer, W. 1975. Modification of E . coli ribosomes and coliphage
MS2 RNA by bisulfite: Effects on ribosomal binding and protein synthesis. Nucleic Acids Res.
2, 501-507.
Bruno, P., Caselli, M., Di Fano, A., and Traini, A. 1979. Fast and simple polarographic method for
determination of free and total sulphur dioxide in wines and other common beverages. Analyst
104, 1083-1087.
Buckley, C. E., Saltzman, H. A., and Sieker, H. 0. 1985. The prevalence and degree of sensitivity
to ingested sulfites. J. Allergy Clin. Immunol. 7 5 , 144.
Buechsenstein, J. W., and Ough, C. S . 1978. SO2 determination by aeration-oxidation: A com-
parison with Ripper. Am. J . Enol. Vitic. 29, 161-164.
Burroughs, L. F. 1975. Determining free sulfur dioxide in red wine. Am. J. Enol. Vitic. 26, 25-29.
Burroughs, L. F. 1977. Stability of patulin to sulfur dioxide and to yeast fermentation. J. Assoc. Of.
Anal. Chem. 60, 100-103.
Burruughs, L. F., and Sparks, A. H. 1973a. Sulphite-binding power of wines and ciders. I. Equi-
librium constants for the dissociation of carbonyl bisulphite compounds. J . Sci. Food Agric. 24,
187- 198.
Burroughs, L. F., and Sparks, A. H. 1973b. Sulphite-binding power of wines and ciders. 11.
Theoretical considerations and calculation of sulphite-binding equilibria. J. Sci. Food Agric. 24,
199-206.
Burroughs, L. F., and Sparks, A. H. 1973c. Sulphite-binding power of wines and ciders. 111.
Determination of carbonyl compounds in a wine and calculation of its sulphite-binding power.
J. Sci. Food Agric. 24, 207-217.
Bush, R. K., Taylor, S . L., and Busse, W. W. 1986. A critical evaluation of clinical trials in reactions
to sulfites. J. Allergy Clin.Immunol. (in press).
Calabrese, E., Sacco, C., Moore, G., and DiNardi, S. 1981. Sulfite oxidase deficiency: A high risk
factor in SO,, sulfite, and bisulfite toxicity? Med. Hypotheses 7 , 133-145.
Cam, J. G., Davies, P. A., and Sparks, A. H. 1976. The toxicity of sulphur dioxide toward certain
lactic acid bacteria from fermented apple juice. J. Appl. Bucreriol. 40, 201-212.
Cecil, R. 1963. Intramolecular bonds in proteins. I. The role of sulfur in proteins. I n “The Proteins”
(H. Neurath, ed.), Vol. 1, 2nd Ed., p. 379. Academic Press, New York.
Chambers, R. W., Aguyagi, S. Y.,Furukawa, Y.,Zawadska, H., and Bhanet, 0. S . 1973. Inactiva-
tion of valine acceptor activity by a cystosine into uracil missense change in the anticodon of
yeast valine transfer RNA. J . Biol. Chem. 248, 5549-5551.
Chandler, B. V., and Clegg, K. M. 1970. Pink discoloration in canned pears. 111. Inhibition by
chemical additives. J . Sci. Food Agric. 21, 323-328.
Chapon, L., Chapon, S., and Djenga, N. 1982. Free sulfite in beers-kinetic study. J. Am. Soc.
Brew. Chem. 40, 31-39.
Chin, S . , Bissell, M. J., and Bassham, J. A. 1977. The consequences of bisulfite exposure in
primary chick embryo fibroblast in culture. Bull. Environ. Conrum. Toxicol. 18, 749-757.
Cluzan, R., Causeret, J., and Hugot, D. 1965. Potassium metabisulfite: Long-term study of toxicity
in the rat. Ann. Biol. Anim. Bioch. Biophys. 5 , 267-281.
Codex Alimentarius Commission. March 1975. Potential daily intake of food additives. Joint
FAO/WHO Food Standards Programme, Codex Committee on Food Additives, 10th session, p.
5 . Report CXlFA 75/5. Food and Agriculture Organization, Rome.
Cohen, H. J., and Fridovich, 1. 1971a. Hepatic sulfite oxidase. Purification and properties. J. Biol.
Chem. 246, 359-366.
Cohen, H.J., and Fridovich, 1. 1971b. Hepatic sulfite oxidase. The nature and function of the heme
prosthetic groups. J. Biol. Chem. 246, 367-373.
Cohen, H.J., Fridovich, I., and Rajagopalan, K. V.1971. Hepatic sulfite oxidase: A functional role
for molybdenum. J. Biol. Chem. 246, 374-382.
Cohen, H. J., Drew, R. T., Johnson, J. L., and Rajagopalan, K. V. 1973. Molecular basis of the
biological function of molybdenum. The relationship between sulfite oxidase and the acute
toxicity of bisulfite and SOz. Proc. Natl. Acad. Sci. U.S.A. 70, 3655-3659.
Cruess, W. V., and Glickson, D. 1932. Observations on brining and candying of citron. Fruir Prod.
J . 12, 17-18, 25.
Daoud, H. N., Luh, B. S., and Miller, M. W. 1977. Effect of blanching, EDTA and NaHS03 on
color and vitamin B6 retention in canned garbanzo beans. J. Food Sci. 42, 375-378.
Das, G., and Runeckles, V. C. 1974. Effect of bisulfite on metabolic development in synchronous
Chorella pyrenoidosa. Environ. Res. 7, 353-362.
Davis, E. G., McBean, D. M., Rooney, M. L., and Gipps, P. G. 1973. Mechanisms of sulphur
dioxide loss from dried fruits in flexible films. J. Food Technol. 8 , 391-405.
Delohery, J . , Castle, W.. Simmul, R., and Allen, D. 1984a. Metabisulfite and SO2 reactivity in
asthmatics. J . Allergy Clin. Immunol. 73, 136.
Delohery, I., Simmul, R., Castle, W. D., and Allen, D. 1984b. The relationship of inhaled sulfur
dioxide reactivity to ingested metabisulfite sensitivity in patients with asthma. Am. Rev. Respir.
Dis. 130, 1027-1032.
Dorange, J.-L., and Dupuy, P. 1972. Mise en evidence d’un action mutaghe de sulfite de sodium
sur la levure. C. R . Acad. Sci. Ser. D 274, 2798-2800.
Don, W., and Truper, H. G. 1976. Sulfite formation by wine yeasts. 111. Properties of sulfite
reductase. Arch. Microbiol. 108, 99-104.
Don, W., Heinzel, M., and Truper, H. G . 1976. Sulfite formation by wine yeasts. I. Relationships
between growth, fermentation, and sulfite formation. Arch. Microbiol. 107, 289-292.
Don, W., Heinzel, M.,and Truper, H. G. 1977. Sulfite formation by wine yeasts. IV. Active uptake
of sulfate by “low” and “high” sulfite producing wine yeasts. Arch. Microbiol. 112, 283-
285.
Doyle, M. P., and Marth, E. H. 1978a. Bisulfite degrades aflatoxin: Effect of temperature and
concentration of bisulfite. J. Food Pror. 41, 774-780.
Doyle, M. P., and Marth, E. H. 1978b. Bisulfite degrades aflatoxin: Effect of citric acid and
methanol and possible mechanisms of degradation. J . Food Pror. 41, 891-896.
Dulak, L., Chaing, G., and Gunnison, A. F. 1984. A sulphite oxidase-deficient rat model: Re-
productive toxicology of sulphite in the female. Food Chem. Toxicol. 22, 599-607.
Duran, K., Korteland, J., Beemer, F. A., Heiden, C. v. d., de Bree, P. K., Brink, M., and
Wadman, S. K. 1979. Variability of sulfituria: Combined deficiency of sulfite oxidase and
xanthine oxidase. In “Models for the Study of Inborn Errors of Metabolism’’ (F. A. Hommes,
ed.), pp. 103-107. Elsevier, Amsterdam.
Eschenbruch, R . 1974. Sulfite and sulfide formation during winemaking-a review. Am. J. Enol.
Viric. 25, 157-161.
Eschenbruch, R., and Bonish, P. 1976. Production of sulphite and sulphide by low- and high-
sulphite forming wine yeasts. Arch. Microbiol. 107, 299-302.
Evelyn, J. 1664. Pomona, or an appendix concerning fruit trees. In relation to cider and several ways
of ordering it. Supplement, “Aphorisms Concerning Cider,” p. 24. Martin & Allestry,
London.
Ezrielev, G. I. 1968. Contribution to the toxicology of sodium metabisulphite. Pharmacol. Toxicol.
31, 120.
Fieger, E. A. 1951. Cause and prevention of black spot on shrimp. Ice Refrig. 120, 49-50.
Firth, R. A., Hill, H. A. 0..Pratt, J. M., Thorp, R. G., and Williams, R. J. P. 1969. The chemistry
of vitamin BIZ. Part XI. Some further formation constants. J. Chem. SOC.A, 381-386.
Fisher, A. A. 1975. Contact dermatitis due to food additives. Curis 16, 691.
Fitzhugh, 0. G.,Knudsen, L. F., and Nelson, A. A. 1946. The chronic toxicity of sulfites. J .
Pharmacol. Exp. Ther. 86, 37-48.
Flaherty, M., Stormont, J. M., and Condemi, J. J. 1985. Metabisulfite (MBS)-associated hepatotox-
icity and protection by cobalamins (B12). J . Allergy Clin. Immunol. 75, 198.
Freedman, B. J. 1977. Asthma induced by sulphur dioxide, benzoate and tartrazine contained in
orange drinks. Clin. Allergy 7, 407-4 15.
Fujita, K.,Ikuzawa, M., Izumi, T., Hamano, T., Mitsuhashi, Y., Matsuki, Y., Adachi, T., Nonogi,
H., Fuke, T., Susuki, H.,Toyoda, M., Ito, Y., and Iwaida, M. 1979. Establishment of a
modified Rankine method for the separate determination of free and combined sulphites in
foods. 2. Lebensm. Unters. Forsch. 168, 206-2 11.
Generoso, W. M., Huff, S. W., and Cain, K. T. 1978. Tests on induction of chromosome aberra-
tions in mouse germ cells with sodium bisulfite. Murar. Res. 56, 363-365.
Gibson, W. B., and Strong, F. M. 1973. Metabolism and elimination of sulphite by rats, mice and
monkeys. Food Costnet. Toxicol. 11, 185-198.
Gibson, W. B . , and Strong, F. M. 1974. Accumulation of ingested sulphite- and sulphate-sulphur
and utilization of sulphited proteins by rats. Food Comer. Toxicol. 12, 625-640.
Gilbert, J . , and McWeeny, D. J. 1976. The reactions of sulphur dioxide in dehydrated vegetables. J .
Sci. Food Agric. 27, 1145- 1146.
Goldfarb, G., and Simon, R. A. 1984. Provocation of sulfite sensitive asthma. J . Allergy Clin.
Immunol. 73, 135.
Green, L. F. 1976. Sulphur dioxide and food preservation-a review. Food Chem. 1, 103-124.
Gunnison, A. F. 1981. Sulphite toxicity: A critical review of in virro and in vivo data. Food Cosmer.
Toxicol. 19, 667-682.
Gunnison, A. F., and Farruggella, T. J. 1979. Preferential S-sulfonate formation in lung and aorta.
Chem.-Biol. Inreracr. 25, 27 1-277.
Gunnison, A. F., and Palmes, E. D. 1973. Persistence of plasma S-sulfonates following exposure of
rabbits to sulfite and sulfur dioxide. Toxicol. Appl. Pharmacol. 24, 266-278.
Gunnison, A. F., and Palmes, E. D. 1974. S-Sulfonates in human plasma following inhalation of
sulfur dioxide. Am. Ind. Hyg. Assoc. J . 35, 288-291.
Gunnison, A. F., and Palmes, E. D. 1976. A model for the metabolism of sulfite in mammals.
Toxicol. Appl. Pharmacol. 38, 1 11-126.
Gunnison, A. F., and Palmes, E. D. 1978. Species variability in plasmas-sulfonate levels during and
following sulfite administration. Chem.-Biol. Interact. 21, 315-329.
Gunnison, A. F., Bresnahan, C. A., and Palmes, E. D. 1977. Comparative sulfite metabolism in the
rat. rabbit, and rhesus monkey. Toxicol. Appl. Pharmacol. 42, 99-109.
Gunnison, A. F., Dulak, L., Chiang, G., Zaccadi, J., and Farruggella, T. J. 1981a. A sulfite
oxidase-deficient rat model: Subchronic toxicology. Food Cosmer. Toxicol. 19, 221-232.
Gunnison, A. F., Farruggella. T. J., Chiang, G., Dulak, L., Zaccardi, J., and Birkner, J. 1981b. A
sulfite-oxidase-deficientrat model: Metabolic characterization. Food Cosmer. Toxicol. 19,209-
220.
Habernicht, H. A., Preuss, L.,and Lovell, R . G. 1983. Sensitivity to ingested metabisulfites: Cause
of brofichospasm and urticaria. Immunol. Allergy Prac. 5 , 243-245.
Hagler, W. M., Jr., Hutchins, J. E., and Hamilton, P. B. 1982. Destruction of aflatoxin in corn with
sodium bisulfite. J. Food Pror. 45, 1287-1291.
Hagler, W. M.,Jr., Hutchins, J. E., and Hamilton, P. B. 1983. Destruction of aflatoxin B, with
sodium bisulfite: Isolation of the major product aflatoxin BIS. J. Food Prof. 46, 295-300.
Haisman, D. R. 1974. The effect of sulphur dioxide on oxidizing enzyme systems in plant tissues. J .
Sci. Food Agric. 25, 803-810.
Hamano, T., Mitsuhashi, Y., Matsuki, Y., Ikuzawa, M., Fujita, K., Izumi, T., Adachi, T., Nonogi,
H., Fuke, T.,Suzuki, H., Toyoda, M., Ito, Y., and Iwaida, M. 1979. Application of gas
chromatography for the separate determination of free and combined sulphites in foods. Z.
Lebensm. Unrers. Forsch. 168, 195-199.
Hayatsu, H. 1976. Bisulfite modification of nucleic acids and their constituents. frog. Nucleic Acid
Res. Mol. Biol. 16, 75-124.
Hayatsu, H., and Miller, R. C., Jr. 1972. The cleavage of DNA by the oxygen-dependent reaction of
bisulfite. Biochem. Biophys. Res. Commun. 46, 120-124.
Hayatsu, H., and Miura, A. 1970. The mutagenic action of sodium bisulfite. Biochem. Biophys. Res.
Commun. 39, 156-160.
Hayatsu, H., Wataya, Y.,Kai, K., and lida, S. 1970. Reaction of sodium bisulfite with uracil,
cytosine, and their derivatives. Biochemistry 9, 2858-2865.
Heinzel, M., and Truper, G. 1976. Sulfite formation by wine yeasts. 11. Properties of ATP-sul-
furylase. Arch. Microbiol. 107, 293-297.
Hickey, R. J., Clelland, R. C., Bowers, E.J., and Boyce, D. E. 1976. Health effects of atmospheric
sulfur dioxide and dietary sulfites. Arch. Environ. Health 31, 108-1 12.
Hoppe, J. O., and Goble, F. C. 1951. The intravenous toxicity of sodium bisulfite. J. Pharmacol.
Exp. Ther. 104, 101-106.
Honvitz, W. (ed.) 1975. “Official Methods of Analysis,” 12th Ed., pp. 336-338. Association of
Official Analytical Chemists, Washington, D.C.
Hotzel, D., Muskat, E., Bitsch, I . , Aign, W., Althoff, J.-D., and Cremer, H.D. 1969. Thiamin-
mangel and Unbedenklichkeit von Sulfit fiir den Menschen. Inr. 2. Viruminforsch. 39, 372-
383.
Howland, W. C., and Simon, R. A. 1985. Restaurant-provoked asthma: Sulfite sensitivity? J .
Allergy Clin. Immunol. 75, 145.
Huang, A. S., and Fraser, W. M. 1984. Are sulfite additives really safe? N . Engl. J. Med. 311,542.
Hysert, D. W., and Momson, N. M. 1976. Sulfate metabolism during fermentation. Am. Soc.Brew.
Chem. J . 34, 25-3 I .
Ingram, M. 1959. Technical aspects of the commercial use of antimicrobial chemicals as food
preservatives. Chem. Ind. 552-557.
Inoue, M., Hayatsu, H.,and Tanooka, H. 1972. Concentration effect of bisulfite on the inactivation
of transforming activity of DNA. Chem.-Biol. Interact. 5 , 85-95.
Inouye, B., Morita, K., Ishida, T.,and Ogata, M. 1980. Cooperative effect of sulfite and vanadium
compounds on lipid peroxidation. Toxicol. Appl. Pharmucol. 53, 101-107.
Institute of Food Technologists Expert Panel on Food Safety & Nutrition. 1975. Sulfites as food
additives. Food Technol. 29, 117-120.
Irreverre, F., Mudd, S. H., Heizer, W.D., and Laster, L. 1967. Sulfite oxidase deficiency: Studies
of a patient with mental retardation, dislocated ocular lenses, and abnormal urinary excretion of
S-sulfo-L-cysteine, sulfite, and thiosulfate. Biochem. Med. 1, 187-216.
Jacobs, D. D. 1976. Effect of dissolved oxygen on free sulfur dioxide in red wines. Am. J. Enol.
Vitic. 27, 42-43.
Jacobsen, D. W., Simon, R. A,, and Singh, M. 1984. Sulfite oxidase deficiency and cobalarnin
protection in sulfite-sensitive asthmatics (SSA). J . Allergy Clin.Immunol. 73, 135.
Jagiello, G . M., Lin, J. S., and Ducayen, M. B. 1975. SOz and its metabolites: Effects on mam-
malian egg chromosomes. Environ. Res. 9, 84-93.
Jaulrnes, P. 1970. Etat actuel des techniques pour &placement de I’anhydride sulfureux. Bull. Off.
Int. Vigne Vin 478, 1333.
Jennings, N., Bunton, N. G., Crosby, N. T., and Alliston, T. G. 1978. A comparison of three
methods for the determination of sulphur dioxide in food and drink. J. Assoc. Publ. Anal. 16,
59-70.
Johnson, J. L., and Rajagopalan, K. V. 1976a. Purification and properties of sulfite oxidase from
human liver. J . Clin. Invesr. 58, 543-550.
Johnson, J. L., and Rajagopalan, K. V. 1976b. Human sulfite oxidase deficiency. Characterization
of the molecular defect in a multicomponent system. J . Clin. Invesr. 58, 551-556.
Johnson, J. L., Rajagopolan, K. V., and Cohen, H. J. 1974. Molecular basis of the biological
function of molybdenum. Effect of tungsten on xanthine oxidase and sulfite oxidase in the rat. J.
Biol. Chem. 249, 859-866.
Johnson, J. L . , Jones, H. P., and Rajagopalan, K. V. 1977. In vitro reconstitution of demolyb-
dosulfite oxidase by a molybdenum cofactor from rat liver and other sources. J. Biol. Chem.
252,4994-5003.
Johnson, J. L., Waud, W. R., Rajagopalan, K. V., Duran, M., Beemer, F. A,, and Wadman, S. K.
1980. Inborn errors of molybdenum metabolism: Combined deficiencies of sulfite oxidase and
xanthine dehydrogenase in a patient lacking the molybdenum cofactor. Proc. Natl. Acad. Sci.
V.S.A. 77, 3715-3719.
Joint FAO/WHO Expert Committee on Food Additives. 1974. Toxicological evaluation of certain
food additives with a review of general principles and of specifications. 17th Report, Food and
Agriculture Organization, Rome.
Joslyn, M. A., and Braverman, J. B. S. 1954. The chemistry and technology of the pretreatment and
preservation of fruit and vegetable products with sulfur dioxide and sulfites. Adv. Food Res. 5,
97-160.
Kaczka, E. A., Wolf, D. E., Kuehl, F. A., and Folkers, K. 1950. Vitamin BIZ:Reactions of cyano-
cobalamin and related compounds. Science 112, 354-355.
Kai, K., TSUNO,T., and Hayatsu, H. 1974. The effect of bisulfite modification on the template
activity of DNA for DNA polymerase I. Nucleic Acids Res. 1, 889-899.
Kaplan, D., McJilton, C., and Luchtel, D. 1975. Bisulfite induced lipid oxidation. Arch. Environ.
Health 30, 507-509.
Kessler, D. L., and Rajagopalan, K. V. 1972. Purification and properties of sulfite oxidase from
chicken liver. J. Biol. Chem. 247, 6566-6573.
Kikigawa, K., and Iizuka, K. 1972. Inhibition of platelet aggregation by bisulfite-sulfite. J. Pharm.
Sci. 61, 1904-1907.
Kingkate, A , , Jaengdawang, C., Chakrangkoon, P., Halilamian, C., and Toyoda, M. 1981. Re-
sidual sulfur dioxide in some Thai noodles. J . Food Pror. 44, 334-336.
Kitamura, N., and Hayatsu, H. 1974. Cleavage of the glycosidic linkage of pyrimidine
ribonucleosides by the bisulfite-oxygen system. Nucleic Acids Res. 1, 75-86.
Kochen, J. 1976. Sulfur dioxide, a respiratory tract irritant, even if ingested. Pediatrics 52, 145-
146.
Koenig, J. Q., Pierson, W. E., and Frank, R. 1980. Acute effects of inhaled SOz plus NaCl droplet
aerosol on pulmonary function in asthmatic adolescents. Environ. Res. 22, 145-153.
Koepke, J. W., Selner, J. C., and Dunhill, A. L. 1983. SO2 derived from bronchodilator solutions.
J . Allergy Clin. Immunol. 71, 147.
Koepke, J. W., Christopher, K. L., Chai, H., and Selner, J. C. 1984a. Dose-dependent bron-
chospasm from sulfites in isoetharine. J . Am. Med. Assoc. 251, 2982-2983.
Koepke, J. W., Selner, J. C., Christopher, K., and Glover, G. 1984b. Inhaled metabisulfite sen-
sitivity. J. Allergy Clin. Immunol. 73, 135.
Komanowsky, M . , Talley, F. B., and Eskew, R. K. 1970. Air drying of cultivated mushrooms.
Food Technol. 24, 80-84.
Kudo, I . , Miura, A., and Hayatsu, H. 1978. Inactivation of bacteriophage X during the aerobic
oxidation of bisulfite. Environ. Res. 16, 205-215.
Kudo, I., Hayatsu, H., Yokoiyama, A., and Kuroda, Y. 1980. Effect of bisulfite on cell-to-
substratum adhesion of Chinese hamster cells in culture. J . Pharmacobiodyn. 3, 74-78.
Lafontaine, A., and Goblet, J. 1955. La toxicit6 des sulfites. Arch. Belg. Med. SOC.Hyg. Med. Trav.
Med. Leg. 13, 281-287.
Lamikanra, 0. 1982. Sulfite-induced oxidation and browning of linolenic acid. J. Food Sci. 47,
2025-2027, 2032.
Lauteaume, M.-Th., Ramel, P., Jaulmes, P., and Manin, D. 1969. Dttermination et comparaison
des DL 50 du mktabisulfite de potassium de l’ethanol et de leur combinaison (hydroxydthane-
sulfonate de potassium) par voie orale sur le rat de souche Wistar. Ann. FalsiJ Expert. Chim.
62, 231-241.
Leuch, 1895. Korresp. bl. Schweiz. Aertze. 609. Cited in Cluzan, R., Causeret, J., and Hugot, D.
1965. Potassium metabisulfite: Long-term study of toxicity in the rat. Ann. Biol. Anim. Bio-
chern. Biophys. 5 , 267-281.
Lewis, R. J., and Tatken, R. L. 1979. “Registry of Toxic Effects of Chemical Substances-1979.’’
U.S. Dept. Health and Human Services, U.S. Govt. Printing Office, Washington, D.C.
Life Sciences Research Office. 1976. Evaluation of the health aspects of sulfiting agents as food
ingredients. Prepared for the Food and Drug Administration under FDA contract No.
223-75-2004, Bethesda, Maryland. 25 pp. \
Life Sciences Research Office. 1985. The reexamination of the GRAS status of sulfiting agents.
Prepared for the Food and Drug Administration under FDA contract No. 223-83-2020, Beth-
esda, Maryland. 96 pp.
Linn, W. S., Shamoo, D. A., Spier, C. E., Valencia, L. M., Anzar, U. T., Venet, T. G., and
Hackney, J . D. 1983. Respiratory effects of 0.75 ppm sulfur dioxide in exercising asthmatics:
Influence of upper-respiratory defenses. Environ. Res. 30, 340-348.
Lisberg, G., and Chen, T . 4 . 1973. Storage stability of dehydrated potato granules packaged in cans
and cartons. J. Food Sci. 38, 363-364.
Liu, J.-W. R., and Gallander, J. F. 1983. Effect of pH and sulfur dioxide on the rate of malolactic
fermentation in red table wines. Am. J . Enol. Viric. 34, 44-46.
Lockett, M. F., and Natoff, I. L. 1960. A study of the toxicity of sulfite. J. Phurm. Phurmacol. 12,
488-496.
Luh, B. S., Karbassi, M., and Schweigert, B. S. 1978. Thiamine, riboflavin, niacin and color
retention in canned small white and garbanzo beans as affected by sulfite treatment. J . Food Sci.
43,431-434.
Lukes, B., O’Brien, T. J., and Scanlan, R. A. 1980. Residual sulfur dioxide in finished malt:
Colorimetric determination and relation to N-nitrosodimethylamine. Am. SOC.Brew. Chem. J .
38, 146-148.
McBean, D. M. 1967. Levels of free and combined sulfur dioxide in fruits during sulfuring and
drying. Food Technol. 21, 1402-1406.
McGinnis, R. A. 1982. “Beet-Sugar Technology,” p. 265. Beet Sugar Development Foundation,
Fort Collins, Colorado.
MacLeod, R. M., Farkas, N., Fridovich, I., and Handler, P. 1961. Purification and properties of
hepatic sulfite oxidase. J . Biol. Chem. 236, 1841-1846.
MacRae, W. D., and Stich, H. F. 1979. Induction of sister chromatid exchanges in Chinese hamster
cells by the reducing agents bisulfite and ascorbic acid. Toxicology 13, 167-174.
McWeeny, D. J . 1979. The chemical behavior of food additives. Proc. Nurr. SOC. 38, 129-133.
McWeeny, D. J., Knowles, M. E., and Hearne, J. F. 1974. The chemistry of non-enzymic browning
in foods and its control by sulphites. J. Sci. Fd. Agric. 25, 735-746.
McWeeny, D. J., Shepard, M. J., and Bates, M. L. 1980. Physical loss and chemical reactions of
SO2 in strawberry jam production. J. Food Technol. 15, 613-617.
Mallon, R. G . , and Rossman, T. B. 1981. Bisulfite (sulfur dioxide) is a comutagen in E . coli and in
Chinese hamster cells. Mutar. Res. 88, 125-133.
Means, G. E., and Feeney, R. E. 1971. “Chemical Modification of Proteins,” p. 152. Holden-Day,
San Francisco.
Meggs, W. J., Atkins, F. M., Wright, R. H., Fishman, M., Kaliner, M. A , , and Metcalfe, D. D.
1985. Sulfite challenges in patients with systemic mastocytosis (SM) or unexplained anaphylax-
is (UEA). J. Allergy Clin. Immunol. 75, 144.
Mitsuhashi, Y.,Hamano, T., Hasegawa, A., Tanaka, K., Matsuki, Y.,Adachi, T., Obara, K.,
Nonogi, H., Fuke, T., Sudo, M., Ikuzawa, M., Fujita, K., Izumi, T., Ogawa, S., Toyoda, M.,
Ito, Y., and Iwaida, M. 1979. Comparative determination of free and combined sulphites in
foods by the modified Rankine method and flame photometric detection gas chromatography. Z.
Lebensm. Vnters. Forsch. 168, 299-304.
Monier-Williams, G. W. 1927. “Determination of Sulphur Dioxide in Foods.” Public Health and
Medical Subjects, Rept. No. 43, pp. 1-56. Great Britain Ministry of Health, London.
Montgomery, M. W, 1983. Cysteine as an inhibitor of browning in pear juice concentrate. J. Food
Sci. 48, 951-952.
Moms, J. R., Cawthon, D. L., and Fleming, J. W. 1979. Effects of temperature and SOz addition on
quality and postharvest behavior of mechanically-harvested juice grapes in Arkansas. J. Am.
SOC. Hortic. Sci. 104, 166-169.
Mudd, S.H., Irreverre, F., and Laster, L. 1967. Sulfite oxidase deficiency in man: Demonstration of
the enzymatic defect. Science 156, 1599-1601.
Mukai, F., Hawryluk, I., and Shapiro, R. 1970. The mutagenic specificity of sodium bisulfite.
Biochem. Biophys. Res. Commun. 39, 983-988.
Miiller, F., and Massey, V. 1969. Flavin-sulfite complexes and their structures. J. Biol. Chem. 244,
4007-4016.
Miinzer, R. 1980. Untersuchungen zur mutagenen Wirkung von Bisulfit. Lebensmittel-Wiss. Tech-
nol. 13, 219-220.
Nadel, J. A., Salem, H., Tamplin, B., and Tokiwa, Y. 1965. Mechanism of bronchoconstriction
during inhalation of sulfur dioxide. J. Appl. Physiol. 20, 164-167.
Nelson, K. E. 1983. Effects of in-package sulfur dioxide generators, package liners, and temperature
on decay and desiccation of table grapes. Am. J. Enol. Vitic. 34, 10-16.
Nelson, K. E.,and Ahmedullah, M. 1973. Effect of temperature change on the release rate of sulfur
dioxide from two-stage sodium bisulfite generators. Am. J . Enol. Viric. 24, 75-80.
Nelson, K. E., and Ahmedullah, M. 1976. Packaging and decay-control systems for storage and
transit of table grapes for export. Am. J. Enof. Vitic. 27, 74-79.
Nir, I., Kafri, I., and Cohen, R. 1978. Effect of pelleting on the vitamin K activity of various vitamin
K3 preparations in chicks. Poultry Sci. 57, 206-209.
Njagi, G. D. E., and Gapalan, H. N. B. 1982. Cytogenetic effects of the food preservatives-
sodium benzoate and sodium sulphite on Viciufubu root meristems. Murut. Res. 102, 213-219.
Nofre, C., Dufour, H., and Cier, A. 1963. Toxicit6 g6nCrale compare6 des anions mintraux chez la
souris. C . R. Acud. Sci. 257, 791-794.
Nury, N. S., Taylor, D. H., and Brekke, J. E. 1959. Modified direct colorimetric method for
determination of sulfur dioxide in dried fruit. Agric. Food Chem. 7, 351-353.
Ogawa, S., Suzuki, H., Toyoda, M., Ito, Y.,Iwaida, M., Nonogi, H., Fuke, T., Obara, K.,
Adachi, T., Fujita, K., Ikuzawa, M., Izumi, T., Hamano, T., Mitsuhashi, Y.,and Matsuki, Y.
1979. Colorimetric microdetermination of sulphites in foods by use of the modified Rankine
apparatus. Z. Lebensm. Unters. Forsch. 168, 293-298.
Ogier, H., Saudubray, J. M., Charpentier, C., Munnich, A., Perignon, J. L., Kesseler, J., and
Frezal, A. 1982. Double deficit en sulfite et xanthine oxydase, cause d’encephalopathie due a
une anomalie hereditaire du metabolisme du molybdene. Ann. Med. Interne 133, 594-596.
Oshino, N., and Chance, B. 1975. The properties of sulfite oxidation in perfused rat liver: Interaction
of sulfite oxidase with the mitochondria1 respiratory chain. Arch. Biochem. Biophys. 170,514-
528.
Pitman, I. H., and Jain, N. B. 1979. Covalent addition of bisulfite ion to N-alkylated uracils and
thiouracils. Aust. J. Chem. 32, 545-552.
Ponting, J. D., and Johnson, G.1945. Determination of sulfur dioxide in fruits. Ind. Eng. Chem. 17,
682-686.
Ponting, J. D., Jackson, R., and Watters, G. 1971. Refrigerated apple slices: Effects of pH, sulfites
and calcium on texture. J. Food Sci. 36, 349-350.
Prenner, B. M., and Stevens, J. J. 1976. Anaphylaxis after ingestion of sodium bisulfite. Ann.
Allergy 37, 180-182.
Rankine, B. C., and Pocock, K. F. 1969. Influence of yeast strain on binding of sulfur dioxide in
wines, and on its formation during fermentation. J. Sci. Food Agric. 20, 104-109.
Rankine, B. C., and Pocock, K. F. 1970. Alkalimetric determination of sulphur dioxide in wine.
Ausr. Wine Brew. Spirir Rev. 29, 40-44.
Reith, J. F., and Willems, J. J. L. 1968. Ober die Bestimmung der schwefligen Saure in Lebensmit-
teln. Z. Lebensm. Unters. Forsch. 108, 270-280.
Renner, H. W., and Wever, J. 1983. Attempts to induce cytogenetic effects with sulphite in sulphite
oxidase-deficient Chinese hamsters and mice. Food Chem. Toxicol. 21, 123-127.
Ripper, M. 1892. Die schweflige Saure im Weine und deren Bestimmung. J. Prakt. Chem. 46,428-
473.
Roberts, A. C., and McWeeny, D. J. 1972. The uses of sulphur dioxide in the food industry-a
review. J. Food Technol. 7, 221-238.
Rork, G. S., and Pitman, I. H. 1974. Elimination of bisulfite ion from a series of uracil-bisulfite
adducts. Evidence for a two-step mechanism. J . Am. Chem. SOC.96, 4654-4663.
Rost, E., and Franz, F. 1913. Vergleichende Untersuchung der phamakologischen Wirkiingen der
organisch gehundenen schwefligen Sauren und des neutralen schwefligsauren Natriums. Arb.
Kaiserl. Gesundh. 43, 187-303.
Rudzki, E. 1979. Two not yet described contact allergens: Sodium hyposulfite and tertiary butyl
alcohol. Przegl. Dermarol. 66, 375-377.
Salo, R. J., and Cliver, D. 0. 1978. Inactivation of enteroviruses by ascorbic acid and sodium
bisulfite. Appl. Environ. Microbiol. 36, 68-75.
Sayavedra, L. A., and Montgomery, M. W. 1983. Effect of moisture, temperature and sulfur dioxide
on color of dried applies. Annu. IFT Meet., 43rd, Abstr. No. 301.
Schroeter, L. C. 1966. “Sulfur Dioxide-Applications in Foods, Beverages, and Pharmaceuticals.”
Pergamon, Oxford.
Schwartz, H. J. 1983. Sensitivity to ingested metabisulfite: Variations in clinical presentation. J .
Allergy Clin. Immunol. 71, 487-489.
Schwartz, H. J. 1984. Observations on the uses and effects of sulfiting agents in foods and drugs.
Immunol. Allergy Prac. 6, 29-34.
Schwartz, H. J., and Chester, E. H. 1984. Bronchospastic responses to aerosolized metabisulfite in
asthmatic subjects: Potential mechanisms and clinical implications. J. Allergy Clin. Immunol.
74,511-513.
Seyal, M. A., Nagy, S. M., Fletcher M. P., Ough, C. S., and Gershwin, M. E. 1984. Response of
asthmatic and normal volunteers to the sulfiting agents of wine. J. Allergy Clin. Immunol. 73,
135.
Shapiro, R. 1977. Genetic effects of bisulfite (sulfur dioxide). Murar. Res. 39, 149-176.
Shapiro, R., and Braverman, B. 1972. Modification of polyuridylic acid by bisulfite: Effect on
double helix formation and coding properties. Biochem. Biophys. Res. Commun. 47,544-550.
Shapiro, R., and Gazit, A. 1977. Crosslinking of nucleic acids and proteins by bisulfite. Adv. Exp.
Med. Biol. 86A, 633-640.
Shapiro, R., Cohen, B. I., and Servis, R. E. 1970a. Specific deamination of RNA by sodium
bisulphite. Nature (London) 227, 1047-1048.
Shapiro, R., Servis, R. E., and Welcher, M. 1970b. Reactions of uracil and cytosine derivatives with
sodium bisulfite. A specific deamination method. J. Am. Chem. SOC.92, 422-424.
Shapiro, R., Braverman, B., Louis, J. B., and Servis, R. E. 1973. Nucleic acid reactivity and
conformation. 11. Reaction of cytosine and uracil with sodium bisulfite. J . Eiol. Chem. 248,
4060-4064.
Shapiro, R., DiFate, V., and Welcher, M. 1974. Deamination of cytosine derivatives by bisulfite.
Mechanism of the reaction. J . Am. Chem. SOC. 96, 906-912.
Shapiro, R., Welcher, M., Nelson, V., and DiFate, V. 1976. Reaction of uracil and thymine
derivatives with sodium bisulfite. Studies on the mechanism and reduction of the adduct.
Biochim. Biophys. Acta 425, 115-124.
Sheppard, D., Wong, W. S., Uehara, C. F., Nadel, J. A., and Boushey, H. A. 1980. Lower
threshold and greater bronchomotor responsiveness of asthmatic subjects to sulfur dioxide. Am.
Rev. Respir. Dis. 122, 873-878.
Shih, N. T., and Petering, D. H. 1973. Model reactions for the study of the interaction of sulfur
dioxide with mammalian organisms. Biochem. Biophys. Res. Commun. 55, 1319- 1325.
Shih, V. E., Abroms, I. F., Johnson, J. L., Carney, M., Mandell, R., Robb, R., Cloherty, J. P., and
Rajagopalan, K. V. 1977. Sulfite oxidase deficiency. Biochemical and clinical investigations of
hereditary metabolic disorder in sulfur metabolism. N . Engl. J. Med. 297, 1022-1028.
Simon, R. A. 1984. Adverse reactions to drug additives. J. Allergy Clin. Immunol. 74, 623-630.
Simon, R. A. 1985. Reactivity to inhaled BronkosolR in sulfite sensitive asthmatics (SSA). J .
Simon, R. A,, Green, L., and Stevenson, D. D. 1982. The incidence of ingested metabisulfite
sensitivity in an asthmatic population. J . Allergy Clin. Immunol. 69, 118.
Simon, R., Goldfarb, G., and Jacobsen, D. W. 1984. Blocking studies in sulfite sensitive asth-
matics. J. Allergy Clin. Imrnunol. 73, 136.
Slae, S., and Shapiro, R. 1978. Deamination of cytidine by bisulphite: Mechanism at neutral pH. J .
Org. Chem. 43, 4197-4201.
Smith, E. L., Ball, S., and Ireland, D. M. 1952. B12 vitamins (cobalamins). 2. Neutral, basic, and
acidic cobalamins. Eiochem. J . 52, 395-400.
Somers, T. C., and Evans, M. E. 1977. Spectral evaluation of young red wines: Anthocyanin
equilibria, total phenolics, free and molecular S02. “chemical age.” J. Sci. Food Agric. 28,
279-287.
Sonin, L . , and Patterson, R. 1985. Metabisulfite challenge in patients with idiopathic anaphylaxis. J.
Allergy Clin. Immunol. 75, 67-69.
Sorbo, B.. and Ohman, S. 1978. Determination of thiosulfate in urine. Scand. J. Clin. Lab. Invesr.
38, 521-527.
Southerland, W. M., Akogyeram, C. 0.. Toghrol, F., Sloan, L., and Schemer, R. 1982. Interaction
of bisulfite with unsaturated fatty acids. J. Toxicol. Environ. Healrh 10, 479-491.
Sprenger, J. D., Altman, L. C., Koenig, J., and Pierson, W. E. 1985. Simultaneous sulfite and
sulfur dioxide (SO2)sensitivity. J. Allergy Clin. Immunol. 75, 144.
Stacey, F. W., and Harris, J. F., Jr. 1963. Formation of carbon-hetero atom bonds by free radical
chain additions to carbon-carbon multiple bonds. In “Organic Reactions” (A. C. Cope, R.
Adams, A. H.Blatt, V. Boekelheide, T. L. Cairns, D. Y.Curtin, and C. Niemann, eds.), Vol.
13, pp. 196-200. Wiley, New York.
Stafford, A. E., Bolin, H. R., and Mackey, B. E. 1972. Absorption of aqueous bisulfite by apricots.
J . Food Sci. 37, 941-943.
Stem, A. C., Wohlers, H. C., Boubel, R. W., and Lowry, W. P. 1973. “Fundamentals of Air
Pollution,” pp. 30-31. Academic Press, New York.
Stevenson, D. D., and Simon, R. A. 1981a. Metabisulfite (K2S2OS) sensitivity in asthmatics. J.
Stevenson, D. D., and Simon, R. A. 1981b. Sensitivity to ingested metabisulfites in asthmatic
subjects. J. Allergy Clin. Immunol. 68, 26-32.
Stevenson, D. D., and Simon, R. A. 1984. Sulfites and asthma. J . Allergy Clin. Immunol. 74,469-
472.
Subcommittee on Review of the GRAS List (Phase 11). 1972. A comprehensive survey of industry on
the use of food chemicals generally recognized as safe (GRAS). Committee on Food Protection,
Division of Biology & Agriculture, National Research Council. National Academy of Sciences,
Washington, D.C.
Summers, G. A,, and Drake, J. W. 1971. Bisulfite mutagens in bacteriophage T4.Genetics 68,603-
607.
Suwa, Y . , Nagao, M., Kosugi, A,, and Sugimura, T. 1982. Sulfite suppresses the mutagenic effect
of coffee. Mutaf. Res. 102, 383-391.
Tanaka, T., Fujii, M., Mori, H., and Hirono, I. 1979. Carcinogenicity test of potassium meta-
bisulfite in mice. Ecotoxicol. Environ. Safety 3, 45 1-453.
Taylor, S. L., Martin, L. B., and Nordlee, J. A. 1985. Detection of sulfite residues in restaurant
salads. 1. Allergy Clin. Immunol. 75, 198.
Thewlis, B. H., and Wade, P. 1974. An investigation into the fate of sulphites added to hard sweet
biscuit doughs. J . Sci. Food Agric. 25, 99-105.
Thompson, J . R., and Pace, D. M. 1962. The effects of sulphur dioxide upon established cell lines
cultivated in vitro. Can. J . Biochem. Physiol. 40,207-217.
Til, H. P., Feron, V. J., and DeGroot, A. P. 1972a. The toxicity of sulphite. I. Long-term feeding
and multigeneration studies in rats. Food Cosmer. Toxicol. 10, 291-310.
Til, H. P., Feron, V. J . , DeGroot, A. P., and Van der Waal, P. 1972b. The toxicity of sulphite. 11.
Short- and long-term feeding studies in pigs. Food Comer. Toxicol. 10, 463-473.
Timson, J. 1973. Action of sodium sulphite on the mitosis of human lymphocytes. Chromosomes
Today 4, 21 1-214.
Towns, S. J., and Mellis, C. M. 1984. Role of acetylsalicylic acid and sodium metabisulfite in
chronic childhood asthma. Pediatrics 73, 631-637.
Turchinsky, M. F., Kusova, K. S., and Budowsky, E. 1. 1974. Conversion of non-covalent interac-
tions in nucleoproteins into covalent bonds: Bisulfite-induced formation of polynucleotide-
protein crosslinks in MS2 bacteriophage virions. FEBS Left. 38, 304-308.
Twarog, F. J. 1983. Metabisulfite sensitivity in asthma: A review. N . Engl. Region Allergy Proc. 4,
100-103.
Twarog, F. J . , and Leung, D. Y. M. 1982. Anaphylaxis to a component of isoetharine (sodium
bisulfite). J. Am. Med. Assoc. 248, 2030-2031.
Vahl, J. M., and Converse, J. F. 1980. Ripper procedure for determining sulfur dioxide in wine: A
collaborative study. J . Assoc. 08.Anal. Chem. 63, 194-199.
Valencia, R., Abrahamson, S., and Wagoner, P. 1972. Testing for sodium bisulfite-induced germ
line mutations in Drosophila melanogasrer. In “Annual Report, Food Research Institute-
1972, Part I. Project Reports,” pp. 108-1 11. University of Wisconsin, Madison.
Vonderschmitt, D. J., Vitols, K. S., Huennekens, F. M., and Scrimgeour, K. G. 1967. Addition of
bisulfite to folate and dihydrofolate. Arch. Biochem. Biophys. 122, 488-493.
Wade, P. 1972. Action of sodium metabisulphite on the properties of hard sweet biscuit dough. J .
Sci. Food Agric. 23, 333-336.
Walbaum, H. 1906. Arch. Hyg. 57, 87. Cited in Cluzan, R., Causeret, J., and Hugot, D. 1965.
Potassium metabisulfite: Long-term study of toxicity in the rat. Ann. Biol. Anim. Biochem.
Biophys. 5, 267-28 I .
Walker, R. 1984. Biological and toxicological consequences of reactions between sulphur(1V) oxo-
anions and food components. Food Chem. 15, 127-138.
Walker, R., Mendoza-Garcia, M. A,, Quattrucci, E., and Zerilli, M. 1983a. Metabolism of 3-
deoxy-4-sulphohexosulose,a reaction product of sulphite in foods, by rat and mouse. Food

Chem. Toxicol. 21, 291-297.
Walker, R., Mendoza-Garcia, M. A., loannides, C., and Quattrucci, E. 1983b.Acute toxicity of 3-
deoxy-4-sulphohexosulose in rats and mice, and in vitro mutagenicity in the Ames test. Food
Chem. Toxicol. 21, 299-303.
Wang, B. C., Hillman, D. E., Speilholz, N. I., andTurndorf, H. 1984.Chronic neurological deficits
and Nescaine-CE-an effect of the anesthetic, 2-chloroprocaine, or the antioxidant, sodium
bisulfite? Anesth. Analg. 63,445-447.
Wang, R. Y.-H., and Ehrlich, M. 1980. Comparison of bisulfite modification of 5-methylde-
oxycytidine and deoxycytidine residues. Fed. Proc.. Fed. Am. Soc. Exp. Eiol. 39, 1882.
Wang, R. Y.-H., Gehrke, C. W., and Ehrlich, M. 1980.Comparison of bisulfite modification of 5-
methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res. 8,4777-4790.
Wedzicha, B. L. 1984.A kinetic model for the sulphite inhibited Maillard reaction. Food Chem. 14,
173-187.
Wedzicha, B. L., and Bindra, N. K. 1980.A rapid method for the determination of sulphur dioxide
in dehydrated vegetables. J . Sci. Food Agric. 31, 286-288.
Wedzicha, B. L.,and Lamikanra, 0. 1983.Sulfite mediated destruction of p-carotene: The partial
characterisation of reaction products. Food Chem. 10, 275-283.
Wedzicha, B. L., and McWeeny, D. J. 1974a.Non-enzymic browning reactions of ascorbic acid and
their inhibition. The production of 3-deoxy-4-sulphopentosulosein mixtures of ascorbic acid,
glycine, and bisulphite ion. J. Sci. Food Agric. 25, 577-587.
Wedzicha, B. L.,and McWeeny, D. J. 1974b.Non-enzymic browning reactions of ascorbic acid and
their inhibition. The identification of 3-deoxy-4-sulphopentosulosein dehydrated, sulphited
cabbage after storage. J. Sci. Food Agric. 25, 589-592.
Wedzicha, B. L., Lamikanra, O., Herrera, J. C., and Panahi, S . 1984. Recent developments in the
understanding of the chemistry of sulphur(1V) oxospecies in dehydrated vegetables. Food
Chem. 15, 141-155.
Weeks, C. 1969. Production of sulfur dioxide-binding compounds and of sulfur dioxide by two
Saccharomyces yeasts. Am. J . Enol. Vitic. 20, 32-39.
Weigand, E. H. 1946.The brined cherry industry. West. Canner Packer 38, 60-63.
Weingartner, K. E., Koburger, J. A., Oblinger, J. L., and Knapp, F. W. 1977.Residual bisulfite in
iced Panaeus shrimp. J. Food Pror. 40, 234-235.
Werth, G. R. 1982. Inhaled metabisulfite sensitivity. J. Allergy Clin. Immunol. 70, 143.
Wesche-Ebeling, P., and Montgomery, M. W. 1983. Extraction and partial characterization of
strawberry polyphenoloxidase. Annu. IFT Meet., 43rd. Abstr. No. 295.
Westley, J. 1980.Rhodanese and the sulfane pool. I n “Enzymatic Basis of Detoxification” (W. B.
Jakoby, ed.), Vol. 11, pp. 245-262. Academic Press, New York.
Whiting, S. J., and Draper, H. H. 1980.The role of sulfate in the calciuria of high protein diets in
adult rats. J. Nutr. 110, 212-222.
Wiley, H. W. 1907. Influence of food preservative and artificial colors on digestion and health.
Sulphurous acid and sulphites. Bur. Chem. U.S. Dept. Agric. Bull. No. 84 (Part Ill).Cited in
Cluzan, R., Causeret, J . , and Hugot, D. 1965. Potassium metabisulfite: Long-term study of
toxicity in the rat. Ann. Eiol. Anim. Biochern. Eiophys. 5 , 267-281.
Wilkins, J. W., Jr., Greene, J. A., Jr., and Weller, J. M. 1968.Toxicity of intraperitoneal bisulfite.
Clin. Pharmacol. Ther. 9, 328-332.
Yang, S. F. 1970. Sulfoxide formation from methionine or its sulfide analogs during aerobic
oxidation of sulfite. Biochemistry 9,5008-5014.
Yang. S. F. 1973. Destruction of tryptophan during the aerobic oxidation of sulfite ions. Environ.
Res. 6, 395-402.
Yang, S. F. 1984. Reactions of oxidation intermediates of sulphite species with some cellular
components of plants. Food Chem. 15, 113-124.
Yang, W. H., Purchase, E. C. R., and Rivington, R. N. 1985. Positive Prausnitz-Kiistner reaction
in metabisulfite sensitive subjects. J . Allergy Clin. Immunol. 75, 198.
Yokoyama, E., Yoder, R. E., and Frank, N. R. 1971. Distribution of 35S in the blood and its
excretion in the urine of dogs exposed to %02.Arch. Environ. Health 22, 389-395.

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SULFITES IN FOODS: USES, ANALYTICAL METHODS,

RESIDUES, FATE, EXPOSURE ASSESSMENT, METABOLISM,

STEVE L. TAYLOR,* NANCY A. HIGLEY,*t

*Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706

asthma were confirmed by positive challenges with capsules or solutions contain-

II. USES OF AND EXPOSURE TO SULFITES IN FOODS

CFR section Subject Sulfiting agents allowed

2lCFR 182.3616 GRAS status KHSO3

sulfiting agents, as outlined in Table 11. Consequently, different treatment levels

Theoretical yield Approximate solubility

Sulfur dioxide so2 100.00 11 at 20°C

Z, From Green (1976).

B. NATURAL OCCURRENCE OF SULFITES IN FOODS

In addition to their use as food additives, it must be remembered that the

C. HISTORY OF USE OF SULFITING AGENTS

Sodium bisulfite 4,900,000

D. CURRENT FOOD APPLICATIONS

1. Overview of Current Applications

2 . Inhibition of Nonenzymatic Browning

Nonenzymatic browning is a term used to describe a family of diverse reac-

3. Inhibition of Various Enzymatic Reactions

A similar reaction occurs in shrimp where enzymatic tyrosine oxidation leads

4. Inhibition and Control of Microorganisms

5. Antioxidant and Reducing Agent Uses

6. Bleaching Agent Uses

7. Use as a Processing Aid

9. Specific Applications and Treatment Levels

of American Societies for Experimental Biology (FASEB) panel attempted to

10. Other Uses for Sulfiting Agents in Foods

11. Additional Benefits of Sulfiting Agents in Foods

Sulfites provide additional benefits in foods beyond those already discussed.

Baked goods Cookies 5 6.2 0.031

Condiments Olives 2 0.1 0.002

Lemon, nonfrozen 800 0.12 0.096

Grain products Corn starch and modified corn 20 3.7 0.074

Dried vegetables Potatoes I5 3.2 0.24

mycotoxins, aflatoxins B, and G,, can be degraded by bisulfite, although rather

E. METHODS FOR MEASUREMENT OF SULFITE RESIDUE LEVELS

1 . Free versus Total SO,

Sulfites exist in foods as sulfurous acid, inorganic sulfites, and a variety of

2 . Analysis of Free SO,

3. Analysis of Total SO,

4. A Critical Appraisal of Sulfite Analysis

F. CHEMISTRY OF SULFITES AND FATE IN FOODS

Sulfur dioxide dissolves readily in water-producing sulfurous acid, H,SO,.

PH Free SO2 (%)

From Green (1976).

2. Fate of Sulfites in Foods

3 . Reactions of Sulfites with Carbonyls

the a,P-unsaturated carbonyl intermediates of the browning reaction (McWeeny

4 . Reactions of Sulfites with Reducing Sugars

5 . Reactions of Sulfites with Proteins and Amino Acids

radical mechanism (Yang, 1970, 1984). Tryptophan can be destroyed by a

6 . Reactions of Sulfites with Vitamins

Sulfites can apparently react with a number of vitamins, including thiamine,

7. Reactions of Sulfites with Nucleic Acids and Nucleotides

8. Reactions of Sulfites with Pigments

9. Reactions of Sulfites with Fatty Acids

Sulfites in the concentration range of 0.5-10 mM can induce the oxidation of

10. Fate of Sulfites in Specific Foods

The proportion of added sulfite existing in the combined form would be

Further evaluation of the fate of [35S]sulfitein dehydrated cabbage and carrots

11. Critical Factors in Determination of the Fate of Sulfites in Foods

G . TREATMENT LEVELS VERSUS RESIDUAL LEVELS