(and potential endogenous formation of N-nitroso compounds)

    First draft prepared by
    Dr G.J.A. Speijers
    Laboratory for Toxicology, National Institute of Public Health
    and Environmental Protection, Bilthoven, Netherlands

    Biological data
         Biochemical aspects
         Absorption, distribution, and excretion
         Formation of N-nitroso compounds
         Effects on enzymes and other biochemical parameters
    Toxicological studies
         Acute toxicity studies
         Short-term toxicity studies
         Long-term toxicity/carcinogenicity studies
         Combined administration of nitrite and nitrosatable
         Reproductive toxicity studies
         Special studies on embryotoxicity/teratogenicity
         Special studies on genotoxicity/mutagenicity
         Genotoxicity studies after combined exposure to
           nitrite and N-nitrosatable precursors
         Special studies on malignant transformation
         Special studies on interaction with antioxidants
         Special studies on effects on vitamin levels
         Observations in humans
              Methaemoglobin formation
              Relationship between nitrate and nitrite intake,
                the subsequent endogenous formation of N-
                nitroso compounds and possible risk of (stomach)
                cancer in humans


         Nitrite was reviewed at the sixth, eighth, seventeenth and
    twentieth meetings of the Committee (Annex 1, references 6, 8, 32
    and 41). At its sixth meeting, the Committee allocated an ADI of
    0-0.4 mg/kg bw to this substance, expressed as sodium nitrite. This
    ADI was based on a marginal reduction in body-weight gain at a dose
    level of 100 mg/kg bw/day in a long-term study in rats. At its
    seventeenth meeting, the Committee lowered the ADI to 0-0.2 mg

    sodium nitrite/kg bw and made it temporary. At that time, the
    Committee used a safety factor higher than normal (500) because a
    marginal effect level was considered and there was a possibility of
    the endogenous formation of N-nitroso compounds from the nitrite
    and N-nitrosatable compounds present together in food and the GI
    tract. At its twentieth meeting, the Committee considered the
    reports of a WHO task group (WHO, 1978) and of a working group of
    the International Agency for Research on Cancer on N-nitroso
    compounds (IARC, 1974) but concluded that they did not provide
    sufficient evidence to revise the temporary status of the ADI.
    Since the previous evaluation of nitrite, numerous toxicological
    and epidemiological data have become available.

         The following monograph summarizes both relevant information
    from the previous monographs and the information that has become
    available since the previous evaluation.


    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

         Nitrite rapidly disappeared from a buffered solution at pH<5,
    simulating gastric conditions, and the rate of disappearance was
    enhanced by the presence of food (Mirvish  et al., 1975).

         Nitrite may also react with gastric contents (e.g.,
    nitrosation) or be reduced by the GI flora. A large part of nitrite
    entering the GI tract may thus disappear before absorption takes
    place (Speijers  et al., 1987).

         Absorption of nitrite from the GI tract of rats was slower
    than that of nitrate. Forty five minutes after intragastric
    instillation of 13N-labelled nitrite, radioactivity was higher in
    the stomach and lower in the liver, kidneys, bladder and
    eviscerated carcass, than in similar experiments with
    13N-labelled nitrate (Witter  et al., 1979).

         Nitrite was not absorbed from the caecum and large intestine
    of rats (Witter & Balish, 1979). Gastric absorption of nitrite was
    noted in labelling and gastric emptying studies using rats and mice
    (Witter  et al., 1979; Mirvish  et al., 1975). In rats, 63% of
    nitrite loss from the stomach was due to emptying and 37% to other
    processes (Mirvish  et al., 1975; Groen, in press). Gastric
    absorption of nitrite seemed faster in mice than in rats.  In vivo,
    the rate of absorption was about 4.5 times greater than the rate of
    chemical degradation (Friedman  et al., 1972).

         Nitrite is normally absent from the body fluids and tissues of
    laboratory animals (Witter & Balish, 1979; Fritsch  et al., 1985).
    The extensive pre-systemic metabolism of nitrite results in an
    absolute bioavailability (i.e. the percentage of the dose reaching
    the systemic circulation) considerably lower than 100% (Groen, in

         Intravenous injections and intratracheal instillation of
    13N-labelled nitrite in mice and rabbits resulted in homogenous
    distribution of radioactivity in the heart, kidneys, liver,
    stomach, intestines, lungs and bladder (ranging from 4.2 to 10.5%)
    within 5 minutes. The 13N was equally distributed in plasma and
    red blood cells with 15-20% of the plasma 13N bound to proteins
    (Parks  et al., 1981).

         Thirty minutes after i.v. injection of nitrite, low levels of
    nitrite were detected in blood and saliva of dogs (Fritsch  et al.,
    1985). Half-lives of nitrite plasma values in the distribution
    phase were 48, 12 and 5 minutes for dogs, sheep and ponies,
    respectively (Schneider & Yeary, 1975).

         Nitrite can cross the placenta of rats and guinea-pigs:
    nitrite injected into pregnant animals appeared after a lag
    of approximately 20 minutes in fetal blood but at a lower
    concentration than in maternal blood, (Grüner  et al., 1973;
    Sinha & Sleight, 1971). Transport of large quantities of nitrite
    into milk is unlikely: nitrite doses inducing methaemoglobinaemia
    in nursing rats did not cause the same adverse effect in sucklings
    (Grüner  et al., 1973).

         Urinary and faecal excretion of nitrite are very low since
    most of the nitrite that enters the bloodstream or passes down the
    GI tract is rapidly converted to nitrate, bound to the GI contents,
    or reduced by enteric bacteria. Nitrite is not secreted in
    significant amounts in saliva or bile (Fritsch  et al., 1985). The
    elimination half-life of nitrite (metabolism plus urinary
    excretion) was 0.5 h in dogs, sheep and ponies (Schneider & Yeary,

         In a balance study in rats, Na15NO2 was administered at a
    level of 1.6% in the diet, as a single or multiple dose. Within 72
    h after administration of the single dose, 68% and 12% of the 15N
    dose was excreted in urine and faeces, respectively. After multiple
    dosing, 59% of the administered 15N was excreted in urine and
    19% in faeces. Some 10% of the dose was excreted in urine as
    15N-nitrite (Wang  et al., 1981). Although this study showed
    that at least 59% of the administered 15N was absorbed from the
    GI tract, it was not clear whether it was absorbed as 15N-nitrite
    or as metabolites (Groen, in press). The important pathway of
    elimination is probably the oxidation of nitrite to nitrate.

         While the main route of excretion of nitrite and metabolites
    is via the urine, some excretion via saliva and bile also occurred
    in dogs. The rapid decline in blood concentrations of nitrite was
    attributed to the reactivity of nitrite with haemoglobin and other
    endogenous compounds, a hypothesis which is supported by the
    increased nitrate level after intravenous administration of nitrite
    (Fritsch  et al., 1985).

         No data concerning the absorption and distribution of nitrite
    in humans have been reported. Indications are that nitrite may be
    absorbed from the GI tract since part of the ingested nitrate is
    converted to nitrite in the oral cavity and stomach (see monograph
    on nitrate), and high MetHb levels in young infants ingesting large
    amounts of nitrate have been reported (Shuval & Gruener, 1972).

         Low levels of nitrite have been detected in the faeces of
    humans on a diet with unknown nitrate and nitrite content.
    Similarly to nitrate, nitrite incubated with fresh faeces under
    anaerobic conditions was rapidly converted by the faecal
    microflora, suggesting that nitrite excretion may well be higher
    than what is actually detected (Archer  et al., 1981;
    Saul  et al., 1981).

    2.1.2  Biotransformation  Animals


         Conventional rats receiving 1000 mg nitrite/litre drinking-
    water had lower nitrite levels in gastric and intestinal tissues
    than rats with a defined gut microflora, whereas the nitrate levels
    were about equal. This indicated a similar uptake of nitrite in both
    groups, but a faster conversion (reduction) rate in conventional rats.
     In vitro experiments confirmed that intestinal bacteria were involved
    in the reduction of nitrite (Balish  et al., 1981; Witter & Balish,


         Absorbed nitrite is rapidly oxidized to nitrate in the blood
    by a mammalian process (Witter & Balish, 1979; Fritsch  et al.,
    1985; Parks  et al., 1981; Boink  et al., in press). The process
    of nitrate generation parallels the methaemoglobin (MetHb)
    formation (Boink  et al., in press; Zeilmaker  et al., in press).
    Intravenous injections of 20 mg/kg bw sodium nitrite in dogs, sheep
    or ponies resulted in nitrate plasma concentrations of 40-100
    mg/litre (0.6-1.6 mmol/l) in all three species within minutes
    (Schneider & Yeary, 1975). Nitrite oxidation to nitrate may also
    occur in the stomach prior to absorption, as demonstrated
     in vitro for mice. However, under  in vivo conditions, nitrite
    is probably absorbed from the stomach before large quantities of
    nitrate are formed (Friedman  et al., 1972).

     Methaemoglobin formation

         Nitrite is involved in the oxidation of haemoglobin (Hb),
    normally present in blood, to methaemoglobin (MetHb). The ferrous
    iron Fe2+ of the haem group is oxidized to ferric iron Fe3+ and
    oxygen and nitrite bind more firmly to this oxidized haem (Jaffé,
    1981). During the process of MetHb formation, nitrate is eventually
    generated from nitrite (Boink  et al., in press). Thus, blood
    oxygen transport to the tissues and organs is inhibited. The rate

    of formation of MetHb varies considerably between animal species.
    Hb solutions from ruminants (sheep, goats, cows) incubated with
    freshly prepared sodium nitrite revealed a faster rate of MetHb
    formation than in humans, horses and pigs. At low levels of nitrite
    exposure, MetHb formation is reversible (Smith & Beutler, 1966).

         The rate of MetHb reduction, which is catalyzed by the enzyme
    system NADPH-MetHb reductase, also varied among species, with a
    high correlation between formation and reduction rates of MetHb
    (Smith & Beutler, 1966). At high nitrite exposure, the reductase
    system becomes saturated and can no longer cope with MetHb
    formation. Such saturation results in increased MetHb concentration
    in the blood leading to ischaemia in tissues, cyanosis,
    irreversible damage to the tissues and ultimately to mortality
    (Boink  et al., in press; Walley & Flanagan, 1987: Kross  et al.,
    1992; Fritsch & de Saint Blanquat, 1992; Dudley & Solomon, 1993).

         Methylene blue has a protective effect against nitrite-induced
    MetHb formation and may therefore be used as an antidote in nitrite
    intoxications (Sheehy & Way, 1974). It is also used to verify
    whether certain toxic effects are mediated by nitrite and MetHb or
    by other compounds. Dietary factors may have a protective effect
    against MetHb formation. Examples are ascorbic acid (vitamin C) and
    methionine which reduced the level of nitrite-induced MetHb when
    added to the diet of guinea-pigs. Conversely, guinea-pigs deficient
    in ascorbic acid had higher nitrite-induced MetHb levels than
    control animals (Speijers  et al., 1987).

         Feeder pigs and piglets given doses of 31-62 mg NO2/kg bw,
    induced a moderate rise of MetHb concentration (maximum 13%),
    indicating that MetHb was not a good quantitative criterion for
    nitrite intake (Dvorak, 1984).

         Studies of ferrihaemoglobin (MetHb) formation by amylnitrite
    and sodium nitrite were conducted  in vivo and  in vitro in
    different animal species including humans. In  in vivo studies,
    amylnitrite was administered i.v., i.m., by inhalation or orally,
    while sodium nitrite was injected i.v. In dogs, cats, rabbits and
    rats, i.v. amylnitrite produced HbFe3+ much more rapidly than
    sodium nitrite. In dogs, i.m. injection of amylnitrite was followed
    by a very slow linear increase in the HbFe3+ content. Inhalation
    of amylnitrite did not lead to HbFe3+ formation in dogs, except
    if breathed in closed systems. In dogs, oral administration of
    amylnitrite produced HbFe3+. Inhalation of amylnitrite by human
    volunteers in a gas mask and from ampoules crushed close to the
    nose did not induce haemoglobin oxidation to any significant

    extent, but was associated with headache, tiredness, dizziness and
    a fall in blood pressure. In  in vitro studies, and in contrast to
    sodium nitrite, amylnitrite produced HbFe3+ instantaneously in
    erythrocytes of various species and in human Hb. Theoretically, one
    mole of amylnitrite yields 2 moles of Fe3+. However, only 20% of
    the Fe+3 released during oxidation of Hb by amylnitrite or sodium
    nitrite was recovered (Klimmek  et al., 1988).  Humans

         Nitrite reduction occurs  in vivo yielding nitric oxide
    (Wishnok, in press; Gangolli  et al., 1994). This process may be
    catalyzed by nutritional factors (Newmark & Mergens, 1981). No data
    were reported concerning the  in vivo oxidation of nitrite in

         Nitrite causes MetHb formation in animals and humans. The rate
    of MetHb formation and reduction in humans is slower than in
    ruminants, but faster than in horses and pigs (Smith & Beutler,

         Young infants (aged < 3 months) are extremely susceptible to
    nitrite-induced MetHb formation because of the presence of fetal
    Hb. Fetal Hb can comprise initially 60-80% of the total Hb,
    decreasing to 20-30% within 3 months. Fetal Hb seems to be more
    easily converted to MetHb. In addition, NADPH-MetHb reductase which
    catalyzes MetHb reduction to Hb is normally deficient in neonatal
    erythrocytes (Ellen & Schuller, 1983; Walley & Flanagan, 1987).

         The development of methaemoglobinaemia was investigated in
    three patients after eating meat contaminated with excessive
    nitrites. In two patients (41-year old woman and her 19-year old
    son), the blood gas analysis showed hypoxia, and the blood samples
    were dark brown. After treatment with 35% oxygen and methylene blue
    the patients improved symptomatically. Methaemoglobin concentration,
    measured at a later stage due to delayed diagnosis, were
    respectively 23% in the woman and 7.7% in the son. The third
    patient revealed a profound hypoxia and dark brown coloured blood.
    In this case, the MetHb concentration was measured immediately and
    found to be 66%. Following treatment with high concentration of
    oxygen and 1% methylene blue the patient recovered consciousness
    and the cyanosis cleared within minutes. The meat which had been
    consumed was analyzed and reported to contain 15 000 mg nitrite/kg
    in the first two cases and 10 000 mg/kg in the third case (Walley
    & Flanagan, 1987).

         Bradberry  et al. (1994) reported a case of methaemo-
    globinaemia caused by the accidental contamination of drinking-water
    with sodium nitrite. The patient had a MetHb concentration of 49%.
    The amount of sodium nitrite ingested was estimated to be 0.7 g.
    Cases of MetHb formation due to inhalation or oral exposure to
    amylnitrite have been reported (Machabert  et al., 1994; Dudley &
    Solomon, 1993).

         The effect of sodium nitrite on subpopulations (young and old)
    of isolated neonatal and adult red blood cells was studied.
    MetHb formation increased with NaNO2 concentration in all
    subpopulations. Red blood cells treated with NaNO2 were less
    fragile. Changes in protein composition occurred after NaNO2
    treatment. The membrane-bound Hb increased with increasing NaNO2
    concentration. When compared with adult red blood cells, neonatal
    red blood cells seemed more susceptible to MetHb formation, to
    decrease in fragility, and to oxidative denaturation of spectrins
    and band-3-proteins. Increased susceptibility of neonatal cells to
    oxidative injury and MetHb formation may contribute to their
    shorter life-span when compared to adult cells (Sharma &
    Premachandra, 1993). This susceptibility may also be related to
    lower MetHb reductase activity in neonatal cells (Speijers  et al.,

    2.1.3.  Formation of N-nitroso compounds  Chemistry of nitrosation

         Nitrite may form nitroso compounds by reaction with a
    nitrosatable compound. Many different amino compounds including
    secondary and tertiary amines, secondary and tertiary amides,
    N-substituted ureas, guanidines, indoles (mainly tryptophan-bound
    in proteins) and urethanes, can act as nitrosatable compounds. In
    the case of amines, amides and ureas, the formed nitroso compounds
    are N-nitrosamines, N-nitrosoamides and N-nitrosoureas. The most
    common nitroso compounds are those derived from secondary amines.
    The rate of formation is often pH dependent and proportional to the
    concentration of unprotonated amine (inversely related to the
    basicity of the amine) and to the concentration of N2O3, and
    hence to the square of the NO2 concentration. An optimum pH in
    the range of 2.5-3.3 is commonly observed for N-nitrosamine
    formation (Mirvish, 1975; Challis, 1981, 1985; Foster  et al.,
    1981; Shephard  et al., 1987) The reaction kinetics is of a first
    and second order (Shephard, in press; Janzowski & Eisenbrand, in

         Many nitrosatable compounds (e.g., some aromatic amines,
    amides and ureas) are too unreactive to combine readily with
    N2O3. They react by another pathway, namely through a direct
    reaction of the neutral substrate with either H2ONO+ or NO+.
    Usually these reactions are quite slow at pH > 3 but become
    progressively faster with increasing acidity (Challis, 1981; Shephard,
    in press).

         Nitrosation occurs especially rapidly with weakly basic
    secondary amines (e.g. morpholine, piperazine, N-methylaniline),
    N-alkylureas, N-alkylcarbamates and aminopyrine. Nitrosation
    occurs relatively slowly with strongly basic amines, such as
    dimethylamine, and simple N-alkylamides. Nitrosation of tertiary
    amines, yielding dialkylnitrosamines and nitrosation of guanidines,
    yielding nitrosocyanamides and nitrosoureas occur relatively slowly
    (Mirvish, 1975). Catalysis of N-nitrosamine formation by
    nucleophilic anions at pH 2-5 has been widely observed. The
    catalytic order is SCN > I > Br > Cl > phosphate or
    carboxylate. Acceleration by SCN- and I- have attracted much
    attention because of their  in vivo relevance: salivary SCN-
    levels are relatively high in smokers and I- is present in
    gastric secretion. Nitrosation of amides and related compounds is
    not catalyzed by nucleophilic anions. Effective inhibition of
    nitrosation requires materials which react readily with and convert
    the nitrosating agent to innocuous products e.g. compounds which
    either reduce HNO2 or NO or bind the NO+ group irreversibly.
    Sulfur dioxide and bisulfite ion, ascorbic acid, tocopherols,
    gallic acid, thiols, several dihydroxy phenols and some synthetic
    and natural antioxidants are inhibitors of nitrosation (Challis,
    1981; Leaf  et al., 1987, 1989; Kyrtopoulos  et al., 1991;
    Bartsch  et al., 1989, 1990;).

         Wang & Wu (1991) demonstrated the endogenous formation of
    N-nitrosomorpholine from precursors (high amounts of nitrite and
    morpholine) by determination in the urine. Chinese tea inhibited
    this N-nitrosation which was attributed to the inhibitory effect of
    polyphenolic compounds and ascorbic acid present in tea. Leaf
     et al. (1987) performed a study with ascorbic acid showing
    that the inhibition of the formation of N-nitrosoproline was not

         Nitrosation can take place under several conditions in many
    food products. Only the endogenous nitrosation by reaction of
    nitrite with nitrosatable compounds, mainly in the stomach, will be
    discussed in this monograph.  Endogenous formation of N-nitroso compounds

         Endogenous nitrosation has been studied  in vitro by
    simulation of gastric conditions with precursors of N-nitroso
    compounds, and  in vivo by examination of the stomach contents or
    saliva at intervals after administration of the precursors, and/or
    by determination of N-nitroso compounds in blood, urine, faeces or
    intestinal contents.

     In vitro gastric simulation studies

         The formation of nitrosopiperidine was demonstrated after
    incubation of gastric juice of rats with nitrite and piperidine
    (Alam  et al., 1971a).

         Stomach contents of rats and guinea-pigs were incubated with
    carbaryl, carbofuran or methylurea and sodium nitrite. Less than 1%
    and 19-37% nitrosation of the amides occurred in the stomach
    contents of rats (pH 4-5) and guinea-pigs (pH 1.2-2.6),
    respectively (Rickard & Dorough, 1984).

         Gastric juice of rats, rabbits, cats, dogs and humans was
    incubated with diethylamine and nitrite. More diethylnitrosamine
    was found in gastric juice of rabbits and humans (pH 1-2) than in
    gastric juice of rats and dogs (pH 4-5 and 7.4, respectively)
    (Sen  et al., 1969).

         Several authors demonstrated the formation of nitroso
    compounds after incubation of human gastric juice with nitrite and
    nitrosatable compounds (dimethylamine, diethylamine, L-proline,
    carbaryl, and a number of drugs) (Sander  et al., 1968;
    Walters  et al., 1979; Ziebarth & Teichmann, 1980; Kubacki &
    Kupryzewski, 1980).

         Kyrtopoulos  et al. (1985a,b) showed the formation of nitroso
    compounds after incubation of fasting human gastric juice with
    different amounts of nitrite at pH 2-7. Nitrosopiperidine was
    detected after incubation of gastric juice of rats with nitrate and
    the cyclic amine piperidine (Alam  et al., 1971b).

         Human saliva was incubated with or without nitrate and nitroso
    compounds (nitrosamides and nitrosamines) determined in the
    incubation mixtures. Positive results (100-500/µg/l as N-nitroso-
    pyrrolidine) were obtained in 14/100 and 11/100 samples after
    incubation with and without nitrate, respectively. Before
    incubation, 7/100 samples showed positive results (Hart & Walters,

         In a few studies, food products were incubated with
    (artificial) human gastric juice and nitrosamines were determined
    in the mixture. Homogenates of milk and cheese at pH 1.0 and 3.0,
    similar to human gastric conditions, were treated with nitrite.
    Volatile nitroso compounds were detected only in the cheese
    homogenate, while non-volatile nitroso compounds were detected both
    in the milk and cheese homogenates (Walters  et al., 1974). In a
    later study, slurries of meals (including fried eggs, bread,
    butter, cheese, biscuits, milk and luncheon meat) were incubated
    with human gastric juice and nitrite at pH 2.0. A mean value of
    6.7 mg N-nitrosopiperidine/kg of food was found after 15 minutes.
    Prolonging the incubation time did not cause a further increase in
    this value (Walters  et al., 1979).

         Groenen  et al. (1982) incubated a large number of food
    products with artificial human saliva and gastric juice and
    determined volatile nitrosamines. Most food products did not form
    volatile nitrosamines. Fish and other seafood products, however,
    contained from < 1 to 44 µg dimethylnitrosamine per 'portion'
    (2.5-250 g). Low levels were found with smoked sausage (0.1 µg/
    250 g) and cinnamon (0.2 µg/0.25 g).

         Walters  et al. (1979) nitrosated a tobacco smoke condensate
    under exhaustive (high NO2- content) and simulated gastric
    conditions. About 880 mg extractable nitroso compounds as
    N-nitrosopyrrolidine/kg condensate were found after exhaustive
    nitrosation, compared with 1.2 mg/kg before nitrosation. Under
    simulated gastric conditions, 12 mg extractable nitroso compounds
    as N-nitrosopyrrolidine/kg condensate were found.

     In vivo nitrosation - detection in stomach contents and saliva

         In several animal studies, nitroso compounds were detected in
    the stomach contents after administration of nitrite and a known
    nitrosatable compound. The following amino compounds were
    nitrosated in the stomach of animals in the presence of nitrite:
    dimethylamine, diethylamine, piperidine, pyrrolidine, piperazine,
    diphenylamine, methylbenzylamine, methylaniline, methylurea,
    ethylurea, dimethylglycine, phenmetrazine, carbaryl, carbofuran,
    trimorphamide, ziram, thiram, and daminozide (Lijinsky, 1981;
    Eisenbrand  et al., 1974; Walker, 1981; Iqbal  et al., 1980;
    Borzsonyi  et al., 1980; Sen  et al., 1974; Rickard & Dorough,

         In a few studies, nitrate and a known nitrosatable compound
    were administered. Alam  et al. (1971b) demonstrated the  in vivo
    formation of nitrosopiperidine in the stomach of rats from nitrate
    and piperidine. At comparable doses, a lesser degree of nitrosation
    occurred with nitrite and piperidine. Nitrosomorpholine was formed

    in the stomach of guinea-pigs when nitrate plus morpholine was
    administered in the diet intragastrically after 24-h fast (Roché &
    Ziebarth, 1987). Nitrosophenmetrazine was not detected in the
    stomach of rats after oral administration of nitrate and
    phenmetrazine (Greenblatt & Mirvish, 1973).

         When methylurea (7.5 µmol) and sodium nitrite (15 µmol) were
    given orally to guinea-pigs, 0.7-1.0 µmol of N-nitrosomethylurea
    (NMU) was detected in the stomach 10 minutes after treatment. NMU
    formed readily in the stomach and was absorbed into the blood
    (Yamamoto  et al., 1987)

         Diphenylnitrosamine was detected in the stomach of 11/31
    gastric patients after intake of sodium bicarbonate, nitrate and
    diphenylamine (Sander & Self 1969). Mononitrosopiperazine was
    detected in gastric juice 30 minutes after oral administration of
    piperazine to 4 fasting male volunteers (Bellander  et al., 1984).

         Volunteers received meals with different nitrate contents viz.
    (i) fish with high-nitrate vegetables (ii) fish with low-nitrate
    vegetables, or (iii) meat or eggs with high-nitrate vegetables. At
    0.5-2 h after consumption, dimethylnitrosamine levels up to 7.6,
    3.7 and 0.9 µg/kg gastric liquid were found for the three meals,
    respectively. In some cases, peaks of 16-30 µg/kg were found 4-5 h
    after consumption of the meal with fish and high-nitrate
    vegetables. A large inter-individual variation in nitrosamine
    formation was observed. In some cases, diethylnitrosamine was
    detected at concentrations of up to 13 µg/kg, 0.53 h after
    consumption of the meal with fish and high-nitrate vegetables.
    Dipropyl- and dibutylnitrosamine, N-nitrosopiperidine, N-nitroso-
    morpholine and N-nitrosopyrrolidine were not detected. In fasting
    gastric juice, < 0.2-0.7 µg dimethylnitrosamine/kg was found
    (Groenen  et al., 1984).

         Homogenates of meals consisting of eggs, milk and luncheon
    meat were administered by oral tube to a volunteer. The stomach
    contents were analyzed for volatile nitrosamines up to 60 minutes
    after ingestion of the homogenates. Trace amounts of N-nitroso-
    piperidine (0.36 µg/kg) were occasionally found after 30 minutes.
    N-Nitrosopyrrolidine and volatile nitrosamines of the simple
    dialkyl type were not detected (Walters  et al., 1979).

         Analysis of samples of gastric juice from healthy individuals
    as well as from patients with morphological changes of the gastric
    mucosa and from patients who had undergone gastric surgery, did not
    contain volatile nitrosamines, although some samples contained
    nitrite (Eisenbrand  et al., 1981).

         Gastric contents of volunteers receiving meals with fish, beef
    or bacon together with spinach and vegetable juice, were examined
    for nitrosamines. No significant increase in nitrosamine content
    was observed (Lakritz  et al., 1982).

         Higher nitrosation was not found in fasting gastric juice of
    patients with chronic atrophic gastritis, and no increase in total
    gastric nitroso compounds was found in duododenal ulcer patients
    after cimetidine treatment (Bartsch  et al., 1984). However,
    Reed  et al. (1984) found a significantly higher amount of
    nitrate-reducing bacteria, nitrite and nitroso compounds in fasting
    gastric juice of patients with partial gastrectomy than in normal

         Ten volunteers on 10 separate days within 10 weeks received 16
    or 64 mg NaNO3 in 250 ml of water (about equal or 4 times the
    recommended WHO guideline value of 50 mg NO3-/litre) (WHO,
    1993). Nitroso compounds (amines and amides) were determined in
    saliva before and 1 h after ingestion of nitrate. Nitrate ingestion
    did not affect the level of nitroso compounds in the saliva (Hart
    & Walters, 1983).

         Dimethylnitrosamine was not detected (< 0.1 µg/l) in saliva
    of 27 volunteers just after breakfast or lunch (Eisenbrand  et al.,

         Bacteria, particularly denitrifiers, are capable of mediating
    the endogenous nitrosation of amines when the pH is too high to
    allow nitrous acid-mediated nitrosation (Suzuki & Mitsuoka, 1984;
    Leach  et al., 1987; Janzowski & Eisenbrand, in press). Under
    these conditions characteristic of gastric diseases, a resident
    bacterial flora develops. Nitrosation-proficient bacteria isolated
    from gastric juice of achlorhydric subjects were found to catalyze
    formation of N-nitrosomorpholine  in vitro, and  in vivo in
    achlorhydric rat stomach (Calmels  et al., 1991). Bacteria
    isolated from nasopharyngal microflora also catalyzed nitrosamine
    formation  in vitro. Thus, in addition to structure and amount of
    ingested precursors, gastric pH is a factor of greatest relevance,
    affecting acid- and bacterially-mediated nitrosation (Janzowski &
    Eisenbrand, in press).

     In vivo nitrosation - detection in blood, urine, faeces and intestinal

         Error-free analysis of biological samples containing < 1 µg
    of nitrosamines/kg is difficult. Therefore early report (up to and
    including 1980) on the occurrence of volatile nitrosamines in human
    blood, urine and faeces may be incorrect (Eisenbrand  et al.,
    1981; Lee  et al., 1981; Fine  et al., 1982; Wagner & Tannenbaum, 1985).


         In several studies, blood of normal human subjects contained
    dimethylnitrosamine levels of 0.1-2.5 µg/litre (Lakritz  et al.,
    1982; Yamamoto  et al., 1987, 1989a) and diethylnitrosamine levels
    of <0.1-0.4 µg/litre (Melikian  et al., 1981).

         An increase in blood dimethylnitrosamine levels of
    0.3-0.4 µg/litre was found in 3 human subjects after consumption of
    a meal with bacon, spinach, bread and beer (Fine  et al., 1977).
    However, Melikian  et al. (1981), administering the same meal but
    without beer, found an increase in blood dimethylnitrosamine levels
    in 2 subjects and a decrease in another subject.

         No or very slight increase in blood nitrosamine level was
    found in human subjects after consumption of nitrate-, nitrite-,
    and/or amine-rich meals (Kowalski  et al., 1980; Yamamoto  et al.,
    1987, 1989a; Lakritz  et al., 1982; Groenen  et al., 1984).
    Dimethyl- or diethylnitrosamine were not detected in blood of 23
    patients ingesting 2.5-9 g of ammonium nitrate daily (Ellen  et al.,


         N-nitroso-bis-2-hydroxypropylamine (BHP) was detected in urine
    of Wistar rats given 1% bis(2-hydroxypropyl)amine mixed in powder
    diet and sodium nitrite in distilled water at concentrations of
    0.3% for 94 weeks, but not in rats receiving either of these
    precursors alone (Yamamoto  et al., 1989b; Konishi  et al.,

         Some formation of N-nitrosoproline occurred in germfree and
    gnotobiotic rats offered proline and nitrate in drinking-water
    (Ward  et al., 1986). However, this nitrosation proceeded more
    readily in conventional rats and could be due to a lower pH or a
    role of the gut microflora.

         Guinea-pigs administered 34 mg/litre sodium nitrate
    (0.4 mmol/litre) and proline or thioproline, excreted 2 µg/litre
    nitrosoproline and 28 µg/litre nitrothioproline in urine,
    while guinea-pigs administered 3.5 mg sodium nitrite/litre
    (0.05 mmol/litre) and proline or thioproline, excreted 0.7 and
    13 µg/litre of nitrosoproline and nitrothioproline, respectively
    (Otsuka  et al., 1992).

         N-nitrosoproline was excreted in the urine of male ferrets
    administered 120 mg Na15NO2/kg bw and orally dosed with
    0.87 mmol [2-2H]proline (Perciballi  et al., 1989).

         Nitrosamines were at times present in the urine of persons
    with urinary tract infections, while traces or no nitrosamines were
    detected in the urine of healthy individuals (Hicks  et al., 1978;
    El-Merzabani  et al., 1979; Eisenbrand  et al., 1981).

         No increase was found in urinary nitrosamine levels of human
    subjects after consumption of meals with fish or beef (source of
    amines), bacon (source of pre-formed nitrosamines), in combination
    with spinach and vegetable juice (source of nitrate/nitrite)
    (Lakritz  et al., 1982).

         Volatile nitrosamines were not detected in the urine of 23
    patients after daily oral ingestion of large amounts of ammonium
    nitrate (2.5-9 g) used in preventing the development of renal
    stones (Ellen  et al., 1982a).

         Mononitrosopiperazine was detected in the urine of human
    volunteers after ingestion of piperazine (Bellander  et al.,
    1985). Dimethylnitrosamine was found in the urine of human
    volunteers after ingestion of aminopyrine, or amidopyrine and
    alcohol-containing beverages and/or high-nitrate vegetables
    (Spiegelhalder & Preussmann, 1984; Spiegelhalder, in press).
    Shuker  et al. (1993) also showed the endogenous formation of

         Ohshima & Bartsch (1981) developed a quantitative method for
    measuring nitrosation of proline in humans. Urinary levels of
    nitrosoproline, a non-carcinogenic and non-mutagenic nitrosamine,
    were measured after ingestion of nitrate and proline. Because
    nitrosoproline is not metabolized to any significant extent,
    urinary excretion was used as a quantitative indicator of
    nitrosation  in vivo. The amount of total nitrosoproline excreted
    in urine was proportional to the proline dose and increased
    exponentially with the nitrate dose. At the highest doses of
    nitrate (325 mg) and proline (500 mg), 17-30 µg of nitrosoproline
    (mean 23 µg) was formed within 24 h. Intake of 260 mg nitrate
    together with 500 mg proline resulted in the formation of about
    10 µg nitrosoproline. At intakes of 195 or 130 mg nitrate together
    with 500 mg proline, about 3 and 2 µg nitrosoproline were formed,
    respectively. At 65 mg nitrate together with 500 mg proline, the
    amount of nitrosoproline formed was negligible. When different
    amounts of proline were given (60-500 mg) together with 325 mg
    nitrate, 3-30 µg nitrosoproline was formed.

         Higher amount of urinary nitrosoproline or other nitroso amino
    acids were not found after ingestion of nitrate and proline by
    patients with chronic atrophic gastritis, than in a control
    experiment with healthy persons. Similarly, cimetidine treatment of
    17 duodenal ulcer patients did not lead to increased urinary
    nitrosoproline levels (Bartsch  et al., 1984)

         Mirvish  et al. (1992) performed a study in which they found
    an association between N-nitrosoproline excretion by rural
    Nebraskans and nitrate in drinking-water. The significance of this
    finding for people drinking high-nitrate water remains to be

         Moller  et al. (1989) studied the excretion of N-nitrosoproline
    in 12-h overnight urine samples after intake of 500 mg of L-proline by
    285 individuals in areas of northern Denmark with large variations in
    nitrate concentrations in drinking-water. They concluded that the crude
    association between nitrate concentration in drinking-water and the
    rate of endogenous nitrosation in individuals was only weakly positive
    and not statistically significant.


         Dimethyl- and diethylnitrosamine were detected in normal human
    faeces at levels up to 1.5 and 13 µg/kg, respectively. Lower levels
    (about 1 µg/kg) of dibutylnitrosamine, nitrosopyrrolidine and
    nitrosomorpholine were also found (Wang  et al., 1981). However,
    in later studies no volatile nitrosamines in faeces of healthy
    individuals or patients could be detected (Archer  et al., 1981;
    Eisenbrand  et al., 1981; Lee  et al., 1981). Suzuki & Mitsuoka
    (1985) found nitrosamines in faeces of Japanese individuals and
    reported an increase in nitrosamine levels after consumption of a
    Western diet. In a later study of the same authors (1985), the
    positive results were ascribed to artefactual generation of
    nitrosamine during analytical procedures.

    Intestinal contents

         Formation of nitrosopiperidine from piperidine and nitrite or
    nitrate occurred  in vivo in the intestinal contents of rats
    (Alam  et al., 1971a,b).

         Suzuki & Mitsuoka (1984) and several authors in earlier
    literature (as cited in Suzuki & Mitsuoka, 1984) reported
    nitrosamine formation by intestinal bacteria. Nitrosamine formation
    from nitrite and a secondary amine by some intestinal bacteria was
    due to enzymatic catalysis.

         In contrast, Mallett  et al. (1985) found no differences in
    urinary nitroso-proline excretion by conventional microflora and
    germ-free rats after ingestion of nitrate and proline, suggesting
    no involvement of intestinal microflora in nitrosation of proline.
    It was recently found that intestinal bacteria do not catalyze
    nitrosation of proline at pH > 6, thus constituting a serious
    draw-back to the N-nitroso-L-proline test, since nitrosation of
    other secondary amines is catalyzed by intestinal bacteria at
    elevated pH levels (Crespi  et al., 1987).  Yield of endogenous N-nitroso compounds

         It is known that nitrite and dietary amines can react in the
    body to form (carcinogenic) nitrosamines, only when both precursors
    are administered concomitantly. However, whether endogenous
    nitrosation occurs under actual food intake conditions in large
    enough amounts to pose a risk to human health is still a
    controversial question. The problem is confounded by the variety
    of nitrosating agents and nitrosation pathways that have been
    discovered, by the instability of many of the nitrosated products,
    and by the sheer number and variety of nitrosable precursors that
    are present in the diet (Shephard, in press). In addition, no
    reliable methods for the detection of non-volatile N-nitroso
    compounds are available at present.

         The yields of N-nitroso compounds (NOC) formed endogenously by
    acid catalysis in the stomach have been estimated from the reaction
    characteristics and the  in vivo nitrosation rates of different
    nitrosatable precursors. These yields were calculated for a 'high'
    (72 /µmol) and 'low' (1.7 /µmol) gastric nitrite burden. The
    effects of catalysts and inhibitors on nitrosation were ignored in
    the calculation model. The resulting estimates of NOC yields span
    8 orders of magnitude. The indole side chains of tryptophan
    residues in protein and peptides appeared to be the most important
    source of endogenous nitroso product, with daily yields ranging
    from 1 to 100 µmol. However, this calculation assumed that all side
    chains of denaturated proteins were accessible to nitrite in the
    stomach, which may overestimate the actual nitroso-indole yield.
    Other precursors estimated to produce sizable amounts of endogenous
    NOC are amide and ureas (1 - 50 nmol/day), and aryl amines and
    peptide N-termini at higher nitrite burden (0.5 - 500 nmol/day). At
    the end of the scale are the yields of primary and secondary
    N-nitrosamines and N-nitrosamino acids (sub-picomole to nanomole
    range) (Shephard, in press). It is difficult to check whether the
    calculated yields are realistic, but a good correlation was found
    between predicted and observed  in vivo yields of the stable,
    non-carcinogenic nitroso product N-nitrosoproline (Shephard, in
    press; Ohshima & Bartsch, 1981; Bartsch  et al., 1984; Tannenbaum,
    1987; Bartsch  et al., 1989, 1990; Shapiro  et al., 1991).
    However, most of the products of endogenous nitrosation are either
    chemically unstable or rapidly metabolized (Shephard, in press).
    Although many of the metastable nitrosated products demonstrate
    appreciable activity (mutagenicity and/or alkylating properties)
     in vitro (Meier  et al., 1990; Shephard  et al., 1993), they
    have thus far eluded direct analysis in biological fluids
    (Shephard, in press). The best check for the calculated yields
    (Shephard, in press) could be measurements of the total NOC

    (analysis of the -NO group) in the stomach (Reed  et al., 1984;
    Bavin  et al., 1982). The calculated yield of endogenous N-nitroso
    compounds in the acidic stomach would be compatible with these
    experimental data. Endogenous N-nitroso compounds yields from
    bacterial- and macrophage-mediated nitrosation are still an open
    question (Shephard, in press).

         In most studies, no increase in gastric NOC or urinary
    N-nitrosoproline concentrations was demonstrated in patients with
    chronic atrophic gastritis (Shephard, in press). However, a 3- to
    7-fold increase in the rate of N-nitroso-proline excretion was
    found in patients with chronic urinary tract infections or liver
    cirrhosis. The excretion of secondary N-nitrosamino acids was in
    the range of 10-100 nmol/day (Bartsch  et al., 1989). On the basis
    of the previous calculations, especially with the 'high' stomach
    nitrite level, the endogenous nitrosation could contribute
    substantially to the total N-nitroso compounds burden (Shepherd, in
    press). However, the major questions surrounding the issues of
    endogenous nitrosation products of indoles and peptides, are the
    precise amounts of arylamines and ureas found in the diet and the
    contribution of bacterial- or macrophage-catalyzed nitrosation to
    the endogenous N-nitroso compound burden.

         Bartsch  et al. (1992) reviewed the endogenous formation of
    N-nitroso compounds and human cancer etiology. It was concluded
    that endogenous NOC-formation, DNA damage and gene mutations in
    humans could occur at various sites of the body such as the stomach
    and chronically infected or inflamed organs. Inhabitants of
    high-risk areas for stomach and oesophagal cancer, patients
    with urinary tract infections (at risk for bladder cancer)
    and Thai subjects infected with liver fluke (at risk for
    cholangiocarcinomas) had significantly higher exposure to
    endogenously formed NOC.

    2.1.4  Effects on enzymes and other biochemical parameters

         The small intestine of Wistar rats was perfused continuously
    with 100 ml of sodium nitrite solution, during which the rat was
    under urethane anaesthesia. The rate of this  in situ perfusion in
    the apparatus-intestine system was 20 ml/min and the perfusion
    lasted 1 h. Sodium nitrite was poorly absorbed (10% of the
    administered dose), but inhibited the activity of Na+/K+-ATPase
    and alkaline phosphatase. It had no effect on the lactic acid
    level, pointing to normal level of oxygen in the intestine, but
    evidently reducing the utilization of oxygen by this tissue. Using
    metabolism inhibitors added to the perfusion fluid (ouabain, sodium
    azide, phenylalanine) and during functional ischaemia of the
    intestine produced by occlusion of the superior mesenteric artery
    during perfusion, it was possible to determine the site and nature

    of the action of sodium nitrite. Nitrite acts on the plasma
    membrane of the enterocytes providing a possibility for producing
    lability of these membranes which is associated with changes in
    transport function. The structure of other membrane lipids such as
    membranes of lysosomes or mitochondria might be changed. An
    interaction with the respiratory chain was found (Grudzinski,

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The oral LD50 for sodium nitrite in rats was 85 mg/kg bw
    (Lehman, 1958), and in mice it varied from 175 to 220 mg sodium
    nitrite/kg bw (Greenberg, 1945; Lehman, 1958). Clinical signs of
    acute intoxication in rodents included vasodilatation, lowering of
    the blood pressure, decrease in vitamin A content in the liver, and
    functional disturbance of the thyroid gland.

         Dogs administered a single dose of 1-2 g sodium nitrite/kg of
    sausage showed an increased respiration and heart rate, changes of
    the ECG, methaemo-globinaemia within 1-2 h, increased concentration
    of sodium, decreased concentration of potassium and increased ASAT
    activity in serum (Annex 1, reference 33).

         A difference in toxicity was noticed in a study in female rats
    in which a single dose was compared with a more continuous
    administration of the same dose. Using methaemoglobinaemia as a
    parameter, doses of 160 mg sodium nitrite/kg bw or 320 mg/kg bw
    divided into smaller doses over time (3 intervals of 15 minutes,
    followed by 4 intervals of 30 minutes) appeared to be less toxic
    than single doses of 40 or 80 mg/kg bw (De Vries, 1983).

         In another study, 2 doses of 100 mg sodium nitrite/kg bw
    administered to rats at 2-hour interval caused a high mortality,
    whereas all animals survived at a 4-hour interval (Druckrey  et al.,
    1963). The difference in toxicity could be related to the
    elimination half-life of MetHb which was reported to be 90 minutes
    in rats (Shuval & Gruener, 1972).

         Single doses of 0, 50, 75, 100, or 150 mg NaNO2/kg bw were
    administered to 45- to 55-day old male Long-Evans Hooded rats
    (18-24 animals/group) to study the acute toxic effects of nitrite
    on behaviour, haematology and histopathology of the brain.
    Behavioural changes were studied 25 minutes after administration of
    NaNO2 (75 mg/kg bw) and the histopathology of the brains was
    performed 24 h after dosing. Severe motor incoordination was
    produced by immersing the animals in water for 10 minutes before
    testing, but not when they received mild foot shocks 10 minutes

    before testing. The MetHb formed after nitrite administration was
    determined after these pretreatments. No changes in MetHb due to
    pretreatment were detected. Evidence of a prolonged effect of
    nitrite on cells in the hippocampal formation was noted which
    resembled changes in other cases of ischemia (Isaacson & Fahey, 1987).

    2.2.2.  Short-term toxicity studies  Mice

         In a 7-day study, groups of 6- or 55-week old mice (5/group)
    were administered by gastric intubation 0 or 110 mg sodium
    nitrite/kg bw. A decrease in the forced running distance,
    abnormalities in the ECG and ultramicroscopic changes of the heart
    muscle were observed (Kinoshita  et al., 1985).

         In a 2-week study, concentrations of 0, 100, 1000, 1500 or
    2000 mg sodium nitrite/litre drinking-water (equivalent to 0, 10,
    100, 150, or 200 mg/kg bw/day) caused decreased motor activity in
    mice (Gruener & Shuval, 1971).

         Groups of male mice (10/group) received drinking-water
    containing concentrations of 0, 100, 1000, 1500, or 2000 mg sodium
    nitrite/litre, equivalent to 0, 10, 100, 150 or 200 mg/kg bw/day.
    Motor activity decreased in treated animals, especially in the
    group receiving the highest dose. Methaemoglobinaemia was also
    observed. According to the authors, the sedating effect in mice was
    not associated with methaemoglobimaemia (Behroozi  et al., 1971).  Rats

         In a 6-week study, rats (10/sex/group) received 0, 0.06,
    0.125, 0.25, 0.5 or 1% sodium nitrite via drinking-water,
    equivalent to 0, 60, 125,250, 500 or 1000 mg/kg bw/day. Moderate
    growth depression was observed at 1000 mg/kg bw. At autopsy, a
    marked change in colour (brown) of the blood and spleen due to
    methaemoglobinaemia was noted at the two highest dose groups. The
    maximum tolerated dose was 0.25% in drinking-water (Maekawa  et al.,

         In a 2-month study, male rats (8/group) received 0, 100, 300
    or 2000 mg sodium nitrite/litre in drinking-water, equivalent to
    0, 10, 30 or 200 mg/kg bw/day. Abnormalities in the EEG were
    observed in the highest dose group and to a lesser extent in the
    other dose groups (Shuval & Gruener, 1972).

         In a 90-day study, groups of 6-week old Wistar derived
    SPF-bred rats (10/sex/group) received KNO2 in drinking-water at
    levels of 0, 100, 300, 1000 or 3000 mg/litre. The potassium
    concentrations in the nitrite solutions were equalized by adding
    KCl up to the K+ level of the 3000 mg KNO2/litre solution. An
    additional group received drinking-water supplemented with KCl
    only, to achieve the same K+ concentration as that found in the
    3000 mg KNO2/litre solution. Body weight, food intake and food
    efficiency were decreased at 3000 mg/l in males, while liquid
    intake was decreased in males given 1000 and 3000 mg/l and in
    females given 3000 mg/l. There was a significant increase in the
    MetHb concentration in animals given 3000 mg/l. No impaired renal
    function was observed in any of the test groups, although the
    relative kidney weight and plasma urea nitrogen level were
    increased at the highest dose. There was a slight decrease in
    plasma alkaline phosphatase activity at 3000 mg/l. A small amount
    of nitrite was present in the saliva of rats receiving 3000 mg
    KNO2/l but there was no evidence of increased mutagenic activity
    in the urine of these rats. Interestingly, hypertrophy of the
    adrenal zona glomerulosa was observed in all KNO2 test groups,
    the incidence and degree being dose-related. At 100 mg KNO2/l the
    hypertrophy was not significantly different from the controls but
    the authors considered this increase to be of biological relevance.
    However, the Committee concluded that the NOEL in this study was
    100 mg KNO2/litre, equivalent to 10 mg KNO2/kg bw/day, or
    5.4 mg/kg bw/day expressed as nitrite ion (Til  et al., 1988).

         A supplementary 90-day oral toxicity study revealed a NOEL of
    50 mg KNO2/l, equivalent to 5 mg KNO2/kg bw/day. In this study
    slight decreases in circulating steroid hormones were observed in
    the high-dose group at week 4, but not at week 13 of the study
    (Til  et al., 1990; Til & Kuper, in press).

         In a 90-day study, male Wistar rats were administered KNO2
    in drinking-water at levels of 3.6, 12 or 36 mmol/l (300, 1000 or
    3000 mg/l KNO2. Control animals received 36 mmol KCl/l and
    another group received 36 mmol KNO3/l (3700 mg/l). Additional
    animals were allocated to the control and the high-nitrite group
    for interval kills at 28 or 56 days, or after a 30-day or a 60-day
    recovery period following the treatment period of 90 days. Special
    emphasis was given to the effect on the adrenal and MetHb
    formation. Slight hypertrophy of the adrenal zona glomerulosa was
    seen after 28 clays in rats exposed to 36 mmol/l KNO2. Longer
    exposure time did not result in progression of this adrenal lesion.
    Exposure during 90 days to lower doses of nitrite showed that the
    incidence and degree of the hypertrophy were dose-related, with a
    NOEL of 3.6 mmol/l, equivalent to 30 mg KNO2/kg bw/day, or
    16 mg/kg bw/day expressed as nitrite ion. Slight focal and diffuse

    hypertrophy were still present in rats exposed to 36 mmol/l after
    a recovery period of 30 days, but disappeared after a 60-day
    recovery period. Exposure during 90 days to 36 mmol/l potassium
    nitrate, which is normally readily formed in the circulation
    following administration of nitrite, did not induce hypertrophy of
    the adrenal zona glomerulosa. MetHb formation occurred during the
    first weeks of exposure to 36 mmol/l KNO2. The MetHb level
    decreased gradually with time, suggesting metabolic adaptation to
    prolonged nitrite exposure. It should be noted that there was a
    3-fold lower sensitivity for hypertrophy of the adrenal zona
    glomerulosa in this study compared with study by Til  et al.
    (1988). The difference was not due to the diet used, but probably
    to strain differences (Boink  et al., in press).

         In a 16-week toxicity study, 1-month old male rats (8/group)
    received 0 or 200 mg sodium nitrite/litre via the drinking-water
    (equivalent to 0 or 20 mg/kg bw/day). The methaemoglobin levels in
    the treated animals ranged from 0.5-3.1%, in comparison with 0-1.2%
    in the control group. A higher incidence of pulmonary lesions was
    noticed in the treated group.

         In a second experiment with 2-month old rats (12 in the
    nitrite and 9 in the control group), sodium nitrite was
    administered at levels of 0 or 2000 mg/litre for 14 months. The
    methaemoglobin levels fluctuated from 1-35% in comparison to 0-1%
    in the control group. Animals receiving nitrite had lower body and
    liver weights, decreased vitamin E levels in serum, and higher red
    blood cells reduced glutathione levels, while the lungs of all
    animals exhibited severe lesions (Chow  et al., 1980).  Rabbits

         In line with the changes of the adrenal zona glomerulosa of
    rats observed after exposure to nitrite, Violante et al. (1973)
    reported changes in urinary steroid excretion of rabbits caused by
    parenteral administration of 10 mg NaNO2/kg bw/day for 18 days.
    The changes consisted of a time-dependent decrease in the urinary
    excretion of 17-hydroxy-, 17-keto- and 17-ketogenic steroid. Oral
    administration of 20 mg NaNO2/kg bw/day for 14 days also caused
    a decreased urinary excretion of 17-hydroxy and 17-ketosteroids.

    2.2.3  Long-term toxicity/carcinogenicity studies  Mice

         In a carcinogenicity study, mice (50/sex/group) received
    drinking-water containing 0, 1000, 2500 or 5000 mg sodium
    nitrite/litre for 18 months, equivalent to 0, 200, 500 or
    1000 mg/kg bw/day. No changes in tumour incidence were observed
    (Inai  et al., 1979).

         In a long-term carcinogenicity study, inbred mice (200/group)
    were administered 0 or 0.2% NaNO2/l in drinking-water. One
    hundred mice were exposed  in utero to 0.2% NaNO2 (during
    pregnancy and suckling) and continued on 0.2% NaNO2 in their
    drinking-water during weanling. Routine histological examination
    revealed that NaNO2 had no apparent effect on CNS tumour
    formation irrespective of the length of exposure. This finding
    contradicted previous suggestive evidence that nitrite may be a
    causative factor in cerebral glioma, since these VM mice are
    especially susceptible to spontaneous glioma formation (Hawkes
     et al., 1992).  Rats

         In a large-scale study sponsored by the US FDA (Newberne,
    1978, 1979), 573 control rats and 1383 treated rats, were
    administered nitrite in the diet or drinking-water at doses of
    0, 25, 50, 100 or 200 mg/kg bw. Some animals were exposed during
    their entire life-span starting 5 days prenatal and others were
    treated from the age of weaning onwards. Two types of diets were
    used: a conventional laboratory animal chow, and a semi-synthetic
    diet based on agar. Newberne (1978, 1979) reported an increased
    incidence of lymphomas in all nitrite-treated groups (10.2% versus
    5.4% in control rats). A Governmental Interagency Working Group,
    however, came to different conclusions based upon examination of
    the same histological preparations. The Group diagnosed only a
    small number of lesions as lymphomas and assessed an incidence of
    approximately 1% in both treated and control groups. This
    discrepancy concerned the differentiation between the lymphomas
    diagnosed by Newberne, and the extra-medullar haemotopoiesis,
    plasmacytosis or histiocytic sarcomas diagnosed by the Interagency
    group. Incidence of other types of tumours were not increased
    (FDA, 1980a,b).

         In a carcinogenicity study, F344 rats (50/sex/group) received
    in drinking-water concentrations of 0, 0.125 or 0.25% sodium
    nitrite for 2 years. No carcinogenic effects were observed. A
    significant decrease in tumour incidence was found in the high-dose
    females as compared to controls. Part of this decrease was
    accounted for by mononuclear cell leukaemia which has a rather high
    spontaneous frequency (about 25%) in this rat strain (Maekawa  et al.,

         In a 2-year toxicity study, groups of male rats (8/group)
    received drinking-water containing 0, 100, 1000, 2000 or 3000 mg
    sodium nitrite/litre. There was no significant differences in
    growth, development, mortality or total haemoglobin levels between
    the control and treated groups. However, the methaemoglobin levels
    in the groups receiving sodium nitrite at 1000, 2000, and

    3000 mg/litre were raised significantly throughout the study and
    averaged 5%, 12% and 22% of total haemoglobin, respectively. The
    main histopathological changes occurred in the lungs and heart.
    Focal degeneration and fibrosis of the heart muscle were observed
    in animals receiving the highest dose of nitrite. The coronary
    arteries were thin and dilated in these aged animals, instead of
    thickened and narrowed as is usually seen at that age. Changes in
    the lungs consisted of dilatation of the bronchi with infiltration
    of lymphocytes and alveolar hyperinflation. These changes were
    observed in rats receiving 1000, 2000 and 3000 mg sodium nitrite/
    litre drinking-water. The NOEL in this study was 100 mg/litre
    sodium nitrite, equivalent to 10 mg sodium nitrite/kg bw/day, or
    6.7 mg/kg bw/day expressed as nitrite ion (Shuval & Gruener, 1972).

         In a report by the US National Academy of Science, 21 studies
    in mice and rats concerning the possible carcinogenicity of nitrite
    were summarized. According to the authors, however, a number of
    these studies did not meet accepted standards for an adequate
    evaluation of carcinogenicity, because of the short duration,
    inappropriate route of administration, or the study not being
    designed to test nitrite. None of the studies reported indicated
    any carcinogenic effect of nitrite (NAS, 1981; Birdsall, 1981).

         In a carcinogenicity study, F344 rats (24/sex/group) received
    2000 mg sodium nitrite/kg of feed (equivalent to 100 mg/kg bw/day)
    or drinking-water (equivalent to 200 mg/kg bw/day). No carcinogenic
    effects were observed. A decrease in monocytic leukaemia (a very
    common spontaneous neoplasm in F344 rats) was observed in the
    treated groups in both sexes. Other types of tumours were not
    increased in the animals fed nitrite in the diet or in drinking-water
    (Lijinsky  et al., 1983).

         In a life-time study, 70 male and 140 female F0 rats -
    divided into 6 groups - all their male and female offspring (F0
    were fed cured meat containing 0, 200, 1000 or 4000 mg sodium
    nitrite/kg. The canned pork meat was mixed in a ratio of 45% with
    a semi-synthetic diet, fed  ad libitum. The daily exposure to
    sodium nitrite was about 0, 5, 25 or 100 mg/kg bw. Reproduction was
    unaffected. No significant increase in tumour incidence were
    observed (Olsen  et al., 1984). This study confirmed previous
    timings of a study in which rats (30/sex/group) were fed a cured
    meat diet containing 0, 200 or 5000 mg sodium nitrite/kg. The
    canned meat was mixed in a weight ratio of 40% with a standard diet
    and fed first  ad libitum and later in rations of about 20 g/day.
    The daily dose of sodium nitrite was about 0, 4 or 100 mg sodium
    nitrite/kg bw (Van Logten  et al., 1972).

         A long-term feeding study was performed in 6-week old male
    Fischer 344 rats. Sodium nitrite was administered, as part of a
    reduced-protein diet, to groups of rats (50/group) at dose levels
    of 0.2 or 0.5% for up to 115 weeks. A control group (20 male rats)
    received the reduced-protein diet only. Body-weight gain was
    decreased in the nitrite-treated groups. In the first week of
    treatment, RBC, PCV and Hb concentrations were reduced. The RBC
    continued to fall for 8 weeks, slowly returning to normal by week
    52. A dose-related reduction was noted in both the incidence and
    time of onset of lymphoma, leukemia and testicular interstitial
    cell tumours. Leukemia was only found in animals with lymphomas,
    indicating an association between the two lesions. Under the
    conditions of the study, NaNO2 was not carcinogenic to rats, but
    rather the incidence of tumours was reduced in a dose-related
    manner, which correlated with similar trends in body weights (Grant
    & Butler, 1989).

    2.2.4  Combined administration of nitrite and nitrosatable
           compounds  Mice

         Greenblatt  et al., (1971) found a significantly increased
    incidence of lung adenomas in mice given orally 1000 mg sodium
    nitrite/litre in drinking-water in combination with 700 mg
    piperazine/kg of feed. Lower nitrite levels (250 and 500 mg/1) in
    combination with 6250 mg piperazine/kg of feed also caused
    significant tumour induction. The lowest nitrite level (50 mg/l)
    in combination with 6250 mg piperazine/kg of feed revealed tumour
    incidence comparable to controls.

         In a carcinogenicity study, nitrite and dibutylamine (DBA)
    were administered to 45-day old male non-inbred Swiss albino mice
    (20 mice/group). The dibutylamine (1000 mg/kg) and sodium nitrite
    (2000 mg/kg) were administered in drinking-water. The effects of
    soybean (30%) or ascorbic acid (5000 mg/kg) were studied in two
    additional groups. Three different treatment periods, 4-6, 7-9 or
    10-12 months were applied. The combined administration of DBA and
    nitrite revealed an increase in the incidence of benign tumours in
    the bladder (40%) and of hepatomas (27%). The protective effect of
    soybean and ascorbic acid, added separately to the diet or
    drinking-water, respectively, was demonstrated by a marked
    reduction in dysplastic features and the absence of tumours in both
    liver and urinary bladder (Mokhtar  et al., 1988).  Rats

         Montesano & Magee (1971) demonstrated the methylation of
    nucleic acids in the stomach, liver and small intestines of rats
    following the combined ingestion of 14C-labelled methylurea and
    sodium nitrite. No methylation was observed after the ingestion of
    14C-labelled methylurea alone.

         Shank & Newberne (1976) found an increased incidence of liver
    cell carcinomas and angiosarcomas in the liver and lungs of rats
    exposed to 1000 mg/litre sodium nitrite in drinking-water and 5 mg
    morpholine/kg of feed. Lower sodium nitrite levels (50 or 5 mg/l)
    in combination with 5-1000 mg morpholine/kg of feed, revealed the
    same tumour incidence as in a control group receiving 1000 mg
    morpholine/kg feed.

         Weisburger  et al. (1980) treated homogenates of mackerel
    fish with sodium nitrite at pH 3.0. The extracts were given by
    stomach tube to Wistar rats 3 times/week for 6 months. Twelve to 18
    months after feeding, adenomas and adenocarcinomas in glandular
    stomach, squamous cell carcinomas in forestomach and adenocarcinomas
    in the small intestine and pancreas were observed.

         Squid contains high levels of naturally occurring amines such
    as dimethylamine (DMA), trimethylamine, and trimethylamine-N-oxide
    (TMAO). The hepatotoxicity and hepatocarcinogenicity of squid with
    or without exogenous nitrite were investigated in rats. Acute
    necrosis including polymorphogenic neutrophil infiltration,
    haemorrhage and cholangiofibrosis were observed in the livers of
    most rats fed squid. Hepatocellular carcinoma was induced in 2/12
    rats (16%) by feeding 10% squid in the diet for 10 months. The
    incidence of hepatocellular carcinoma was increased to 4/10 rats
    (33%) when 0.3% NaNO2 was added to the diet. At the end of the
    experiment a marked elevation of serum gamma-GT was observed in the
    nitrite treatment group (ALAT and ASAT were not changed). The
    concentration of DMA in squid was estimated to be 0.19%; this
    concentration did not induce hepatocellular carcinoma under the
    experimental conditions used. It was therefore suggested that
    another major naturally occurring amine in squid, TMAO, could
    be an important factor in the induction of hepatotoxicity and
    hepatocarcinogenicity (Lin & Ho, 1992). [The number of animals used
    were too limited to allow any conclusion to be drawn].

         In a carcinogenicity study, male Wistar rats were administered
    bis-(2-hydroxypropyl) amine (BHPA) mixed in powder diet at a
    concentration of 1%, and sodium nitrite dissolved in distilled
    water at concentrations of 0.15% or 0.3% for 94 weeks. Urinary
    excretion of N-nitrosobis-(2-hydroxypropypl)amine (BHP),
    0.9-1.5 µmol, was detected in rats given 1% BHPA and 0.3% NaNO2

    but not in the groups receiving either one of these precursors
    alone. Nasal cavity, lung, oesophagus, liver and urinary bladder
    tumours were found in animals treated with combinations of 1% BHPA
    and 0.15% or 0.3% NaNO2, suggesting that the target organs were
    similar to those affected when the carcinogen was administered
    exogenously. The incidence of nasal cavity, lung tumours and
    oesophagus tumours reached 74, 58 and 11%, respectively, in rats
    given 1% BHPA and 0.3% NaNO2. The incidence of other tumours was
    not increased (Yamamoto  et al., 1989b).

         In a long-term study, the effects on carcinogenesis of
    combined treatment with sodium ascorbate (NaAsA) or ascorbic acid
    (AsA) and NaNO2, with or without N-methyl-N'-nitro-N-
    nitrosoguanidine (MNNG) pre-treatment, were examined. Groups of 20
    or 15 F344 male rats (6-week old) were given a single intra-gastric
    administration of 150 mg/kg bw MNNG in DMSO:water (1:1) or vehicle
    alone. One week later, the animals received supplements of 1% NaAsA
    or 1% AsA in the diet and 0.3% NaNO2 in drinking-water, alone or
    in combination, or basal diet, until the end of week 52. In
    MNNG-treated rats, the incidence of forestomach papillomas and
    carcinomas were significantly higher than in the group receiving
    NaNO2 alone (84% and 47%, respectively), or the basal diet (30%
    and 10%). Significant increase in carcinomas occurred in the group
    receiving the NaAsA (79%) or AsA (85%) supplements. Without MNNG
    treatment, all animals in the NANO2 group demonstrated mild
    hyperplasia. Additional administration of NaAsA or AsA remarkably
    enhanced the grade of hyperplasia, and resulted in 53% and
    20% incidence of papillomas, respectively. It was therefore
    demonstrated that NaNO2 exerted a promoter action on forestomach
    carcinogenesis with NaAsA and AsA acting as co-promoters. The
    results indicated that combined treatment with NaAsA or AsA and
    NaNO2 may in the long-term promote forestomach carcinomas
    (Yoshida  et al., 1994).  Mice and rats

         After oral ingestion of high doses of sodium nitrite together
    with secondary (dimethylamine, methylbenzylamine) or tertiary
    amines (aminopyrine), toxic effects characteristic of nitrosamines
    were observed in mice and rats (progressive inertia, anorexia,
    ascites, weight loss, mortality, hepatic necrosis). Administration
    of sodium nitrite or the amine alone did not cause such effects
    (Asahina  et al., 1971; Astill & Mulligan, 1977; Lyjinski &
    Greenblatt, 1972).

         Carcinogenicity studies in mice and rats receiving orally a
    nitrosatable compound and nitrite, demonstrated the induction of
    tumours characteristic of the corresponding nitroso compound.
    Amines and amides causing tumour induction in these studies
    included amidopyrine, heptamethyleneamine, oxytetracycline,

    morpholine, N-methylbenzylamine, N-methylaniline, N-methylcyclo-
    hexylamine, imidazolidinone, ethylurea, methylurea, N,N-di-
    methylurea, N-methyl-N'-nitroguanidine, piperazine, N-6-methyl-
    adenosine, and disulfiram. Negative results were obtained with
    dimethyl- and diethylamine in mice and rats, respectively. High
    doses of nitrite and amines or amides were used in these studies.
    These doses were extremely high in comparison to normal human
    exposure conditions (Lijinsky, 1981; Preussmann & Stewart, 1984).

    2.2.5  Reproductive toxicity studies  Rats

         Pregnant rats (12/group) were given sodium nitrite in
    drinking-water at concentrations of 0, 2000 or 3000 mg/litre,
    equivalent to 0, 200 or 300 mg/kg bw/day. Non-pregnant females were
    similarly treated. Pregnant rats developed anaemia and had higher
    methaemoglobin levels than non-pregnant rats receiving similar
    doses. There was a pronounced increase in mortality among the
    newborn rats of treated dams compared with those of untreated
    controls, particularly in the 3-week period before weaning.
    Mortality of the offspring was 6% in controls, 30% at 2000 mg/litre
    and 53% at 3000 mg/litre. Birthweights were similar in all groups
    but growth was markedly reduced in pups of treated dams (Shuval &
    Gruener, 1972).

         Pregnant rats given single doses of sodium nitrite varying
    from 2.5-50 mg/kg bw showed transplacental passage of the chemical
    with the production of methaemoglobin in the fetuses (Shuval &
    Greener, 1972).

         In a 2-generation reproductive toxicity study, groups of rats
    were fed from the time of conception a diet containing 0, 240 or
    460 mg sodium nitrite/kg of feed for 28 months (equivalent to 0, 12
    or 23 mg sodium nitrite/kg bw/day). No effects were observed on
    litter size, postnatal mortality, growth rate or life span (Shank
    & Newberne, 1976).

         Pregnant Long-Evans rats were maintained throughout gestation
    on 0.5, 1, 2 or 3 g NaNO2/litre of drinking-water. There were no
    significant differences between treated and control litters at
    birth. Thereafter, pups of treated dams on 2 and 3 g NaNO2/l
    gained less weight, progressively became severely anaemic and began
    to die by the third week postpartum. By the second week postpartum,
    Hb levels, RBC and MCV of these pups were all drastically reduced
    compared to controls. Fatty liver degeneration were noted and blood
    smears showed marked anisocytosis, hypochromasia and gross chylous
    serum lipemia. Histopathology demonstrated cytoplasmic vacuolization
    of centrilobular hepatocytes and decreased hematopoiesis in bone
    marrow and spleen. Administration of 1 g NaNO2/l resulted in
    haematological effects, but did not affect growth or mortality.

    The dose level of 0.5 g NaNO2/l was at or near the NOEL.
    Cross-fostering indicated that treatment during the lactation
    period was more instrumental in producing lesions than treatment
    during the gestation period (Roth  et al., 1987).

         Neonatal Long-Evans rats from dams receiving 2 or 3 g
    NaNO2/litre in the drinking-water through gestation and lactation
    suffered severe microcytic anaemia as well as growth retardation
    and high mortality. Lipemia, fatty liver damage, decreased
    erythropoiesis of spleen and bone marrow, and reduced plasma and
    tissue iron levels were noted in the affected pups. These effects
    were all consistent with and characteristic of iron deficiency.
    Administration of exogenous iron supplement to pups of treated
    mothers reversed the anaemia and other effects of nitrite toxicity
    noted in both previous studies (Shuval & Gruener, 1972;
    Roth  et al., 1987) and in unsupplemented litter mates. Mothers of
    affected pups were themselves anaemic. Reduced iron content was
    measured in milk of nitrite-treated mothers, and severe iron
    deficiency was recorded in pups. Nitrite-consuming dams thus
    appeared to have a reduced capacity to transfer iron to their pups,
    and the nitrite-associated toxicity in pups was actually the result
    of iron deficiency (Roth & Smith, 1988).  Guinea-pigs

         Guinea-pigs (the number of female animals in each group are
    indicated in parenthesis) were administered potassium nitrite in
    drinking-water at concentrations of 0 (4), 300 (3), 1000 (3), 2000
    (3), 3000 (3), 4000 (6), 5000 (4) or 10 000 (3) mg/litre, equal to
    0, 110, 270, 940, 1110, 1190, 1490 or 3520 mg/kg bw/day, for
    100-240 days. At least one male was present in each cage.
    Methaemoglobin levels were measured. In the 5000 and 10 000 mg
    potassium nitrite/litre dose groups, 100% fetal mortality was
    recorded and one of the females died. At the highest dose level,
    growth inhibition in the maternal guinea-pigs was observed. In
    animals with aborted, mummified or resorbed fetuses, inflammatory
    lesions of the uterus and cervix as well as degenerative lesions of
    the placenta were noticed. No fetal mortality was observed at the
    lower dose levels (Sleight & Atallah, 1968).

         Four pregnant guinea-pigs/group received doses of 0, 50 or
    60 mg sodium nitrite/kg bw/day by subcutaneous injection during the
    last 15 days of pregnancy. In the 50 mg/kg bw/day group the partus
    was normal, while 1 h after administration of 60 mg/kg bw/day fetal
    death followed by abortion occurred in 3 animals. At the time of
    death, maternal and fetal blood methaemoglobin concentration had
    reached peak levels and oxygen pressure was lower in fetal blood
    than in the control animals. The dams in the 60 mg/kg bw/day group
    died within 1 h.

         In a second study, pregnant guinea-pigs (9/group) were given
    a single dose of 0 or 60 mg sodium nitrite/kg bw subcutaneously at
    the end of pregnancy. The dams were sacrificed at intervals of
    0.25-56 h after the nitrite injection. Death occurred in 96% of the
    fetuses at 3 or more hours after nitrite administration. A relative
    narrow range was found between doses of sodium nitrite which had no
    effect on reproduction, doses that killed the fetuses and doses
    that killed the dams (Sinha & Sleight, 1971).  Cattle

         Pregnant cows received by infusion for 30 minutes 7, 9.5 or
    12 mg NO2-/kg bw. Treatment with nitrite resulted in a dose-related
    conversion of maternal Hb into MetHb, a 30-50% decrease in mean
    arterial blood pressure, an increase in heart rate with dose-related
    recovery periods, and a decrease of partial oxygen tension (pO2)
    of maternal blood. Fetal changes included a small increase in MetHb
    content, variable changes in heart rate (tachycardia and bradycardia),
    and decreases in fetal pO2, with considerable differences between
    animals. All calves were born alive. Three cows calved early, 2-3 days
    after the highest nitrite dose. The haematological and cardiovascular
    data suggest that these 3 fetuses experienced a more serious hypoxemic
    stress than the other fetuses (Van't Klooster  et al., 1990).

    2.2.6  Special studies on embryotoxicity/teratogenicity  Mice

         Pregnant ICR-mice (approximately 15 animals/group) were given
    drinking-water containing NaNO2 at concentrations of 0, 100 or
    1000 mg/litre on days 7-18 of gestation. There were no significant
    differences between treated and control groups in parameters
    indicative of developmental toxicity, such as litter size, fetal
    weight, and number of resorbed or dead fetuses. The incidence of
    external and skeletal malformations in fetuses of treated groups
    were not significantly different from those in the controls. No
    significant increase was observed in the frequency of gaps and
    breaks in liver cell chromosomes in fetuses exposed  in utero to
    NaNO2. Teratogenic and mutagenic effects of NaNO2 were absent
    in mice at the doses used (Shimaria  et al., 1989).  Rats

         Rats, 40-day old, were fed heat-sterilized meat containing
    sodium nitrite at levels of 0, 200, 1000 or 4000 mg/kg of feed from
    day 40 onwards (equivalent to 0, 10, 50 or 200 mg/kg bw/day). The
    F1b generation was killed at day 21 of pregnancy. Fertility

    index, number of pre-implantation losses, resorptions and
    malformations were not affected by nitrite treatment. No
    differences in litter size, sex ratio, or average weight of pups
    were observed between controls and treated groups (Carstensen &
    Hasselager, 1972, abstract only).

         In an experiment with 2 groups of 10 and 15 pregnant rats,
    sodium nitrite was administered on days 9 and 10 of pregnancy via
    the diet at concentrations of 3 or 10 g/kg diet, equivalent to 150
    or 500 mg/kg bw/day. No embryotoxic or teratogenic effects were
    induced (Alexandrov  et al., 1990).

    2.2.7  Special studies on genotoxicity/mutagenicity

          In vitro exposure of purified DNA to nitrous acid led to
    mutagenic activity as measured by the formation of lethal mutations
    in nitrous acid-treated DNA transformed to  Bacillus subtilis
    (Strack  et al., 1964; Bresler  et al., 1968). Mutagenic activity
    of nitrite (or nitrous acid) has been reported in bacterial systems
    such as  Escherichia coli and several  Salmonella typhimurium
    strains (Kaudewitz, 1959; Verly  et al., 1967; Brams  et al.,
    1987; Hayashi  et al., 1988; Prival  et al., 1991: Balimandawa
     et al., 1994). Positive results in mutagenic studies have been
    reported with various fungi such as  Aspergillus species and
     Neurospora crassa (WHO, 1978; De Serres  et al., 1967), yeast
     (Saccharomyces cerevisiae), tobacco mosaic virus and bacteriophage T4
    (Strack  et al., 1964; WHO, 1978). Although sodium nitrite showed
    mutagenic effects in the Ames test with different  Salmonella
     typhimurium strains, it was negative in the commercial available
    SOS-chromotest, as were many other mutagens (Brams  et al., 1987).
    According to Nakamura  et al. (1987) sodium nitrite was weakly
    genotoxic in the SOS-chromotest.

         In a study with mouse cells, sodium nitrite without metabolic
    activation did not lead to an increase in single strand breaks,
    but a dose-related increase in gene mutations and chromosome
    aberrations was found at relatively high doses. According to the
    authors, the mutagenic activity was probably due to deamination of
    DNA and not to nitrosamine formation, since nitro-sodimethylamine
    without metabolic activation did not change the mutation frequency
    to any significant extent (Kodama  et al., 1976). Sodium nitrite
    administered in an acid environment (pH about 5), induced an
    increase in 6-TG mutants in V79 hamster cells  in vitro (Budayova,
    1985). Chromosome aberrations were significantly increased in
    cultured hamster cells (Tsuda  et al., 1976). Endo-reduplication
    has also been reported (Tsuda & Kato, 1977). Sodium nitrite induced
    a sharp increase in "aberrant cells" obtained from human embryonic
    lung tissue (Stanford Research Institute, 1972 - report not

    available). In a mouse lymphoma L5178Y thymidine kinase locus
    assay, sodium nitrite was positive at concentrations ranging from
    0.02-1 mmol/litre, indicating a relatively weak response in
    comparison with known mutagenic and carcinogenic compounds
    (Wangenheim & Bolcsfoldi, 1988)

         Syrian hamsters were administered orally sodium nitrite on day
    11 or 12 of gestation. An increase in drug-resistant mutations
    (8-AG and ouabain) was found in cells cultured from hamsters
    embryos. In addition, a dose-dependent increase in micronucleus
    formation was found, although no increased number of chromosome
    aberrations was detected (Inui  et al., 1979). It is possible that
    nitrite not only acted on nucleic acids, but also on proteins or
    -SH compounds, so that the mitotic apparatus, i.e. spindle-fibre
    formation, was also affected and damaged. This could be an
    explanation for the large number of cells with micronuclei in
    contrast with the lack of chromosome abnormalities.

          In vitro morphological transformation of hamster cells by
    sodium nitrite was reported (Tsuda  et al., 1973). Transformation
    in embryonic cells occurred  in vitro, while  in vivo implantation
    of the transformed cells led to neoplasms (Inui  et al., 1979).

         In a  Drosophila wing spot test, Graf  et al. (1989)
    observed a mutagenic effect through changes in frequencies of small
    single and large single spots in the wings somatic cells of

         No mutagenic activity was found in two  in vivo tests, a
    host-mediated assay with the  E. coli K 12 uvr B/rec A DNA repair
    and a micronucleus test with mice (Couchhell & Friedman, 1975; Hayashi
     et al., 1981, 1988; Hellmer & Bolcsfoldi, 1992). Administration of
    about 210 mg sodium nitrite/kg bw in drinking-water to nonpregnant or
    5-18 days pregnant rats, however, induced chromosome aberrations in
    bone marrow of both non-pregnant and pregnant animals as well as in
    the embryonic liver. The ratio of the number of metaphases with
    aberrations in treated and control animals, was higher for embryonic
    liver in comparison to adult bone marrow. This higher incidence may
    have resulted from higher numbers of cells in mitosis by shorter cell
    cycle times in embryonic tissues (El Nahas  et al., 1984; Luca  et al.,

         No significant effects were found on metaphase chromosomes of
    bone marrow of adult rats. Experimental data such as dose levels
    were not known as the report was not available (Stanford Research
    Institute, 1972).

         According to Zimmermann (1977), nitrite may exhibit mutagenic
    activity by three mechanisms: (i) nitrite may deaminate DNA-bases
    in single strand vital DNA. Spontaneous deaminations, however, are
    frequent and DNA-repair systems correcting these lesions are
    present in bacteria and probably mammalian cells as well, (ii)
    formation of intra- or interstrand crosslinks between purine
    residues may occur resulting in distortion of the helix in the case
    of double-stranded DNA. An induction of this type of lesions may be
    enhanced by the presence of molecules proximate to DNA, like
    polyamines, glycols, alcohols and phenols (Thomas  et al., 1979),
    and (iii) nitrite may react with nitrosatable agents to form
    N-nitroso compounds and thus indirectly exhibit mutagenic (and
    carcinogenic) activity.

         Alavantic  et al. (1988) studied the effect of nitrite and
    nitrate  in vivo on germ cells of male mice. UDS (17 days after
    treatment) and sperm abnormality (11 or 17 days after treatment) of
    spermatids were studied after treating mice with doses of 60 or
    120 mg/kg bw/day of nitrite for 17 days (for nitrate the doses were
    600 or 1200 mg/kg bw/day for 3 days). Nitrite (and nitrate) did not
    induce UDS response. The only positive result in the sperm-head
    abnormality test was obtained at a dose of 120 mg/kg bw/day at 11
    and 17 days after treatment. The results were in agreement with
    those of earlier experiments with nitrite (and nitrate) by the same
    authors, suggesting a mutagenic action on the tested germ-cell
    stages of male mice.  Genotoxicity studies after combined exposure to nitrite
             and N-nitrosatable precursors

         Nitrosation products of several drugs (by treatment with
    NaNO2 in acid medium) were shown to possess mutagenic activity in
    a bacterial assay with  Salmonella typhimurium (Andrews  et al.,

         Treatment of some food products (fish, beans, borscht) with
    sodium nitrite (1000 and 5000 mg/kg) at pH 3.0 led to the
    development of mutagenic activity in  S. typhimurium in the
    presence and absence of a metabolic activation system. Mutagenic
    activity of nitrosation products of Japanese foodstuffs (after
    treatment with nitrite at pH 4.2) were detected in  S. typhimurium,
    with and without metabolic activation (Marquardt  et al., 1977;
    Weisburger  et al., 1980).

         Inui  et al. (1978; 1980) administered orally sodium nitrite
    and morpholine or amidopyrine to pregnant Syrian hamsters. Gene
    mutations were found in cultured embryonic cells, most probably due
    to transplacental activity of the nitrosamine formed in the mother.

         In intra-sanguineous host-mediated assays with mice, combined
    administration of nitrite and dimethylamine, morpholine or
    aminopyrine, induced mutagenic activity in the test organisms
     S. typhimurium, in the case of dimethylamine and morpholine, and
    in  Schizosaccharomyces pombe in the case of aminopyrine (Barale
     et al., 1981; Edwards  et al., 1979; Whong  et al., 1979a,b).

         Brambilla (1985) showed that nitrosation products of several
    drugs (after treatment with nitrite in acidic medium) caused DNA
    fragmentation in Chinese hamster ovary cells  in vitro.

         UDS was determined in leucocytes of 10 human subjects after
    the consumption of meals containing varying amounts of nitrate,
    nitrite or nitrosamines. In 6/10 subjects, UDS was significantly
    increased but no correlation could be found with dietary nitrate,
    nitrite or nitrosamine levels or with blood nitrosamine levels
    (Kowalski  et al., 1980). In addition, Miller (1984) did not
    observe any effect of ingested nitrate/nitrite (from lettuce) on
    UDS in leucocytes of human subjects after consumption of an
    amine-containing meal (fish).

    2.2.8  Special studies on malignant transformation

         The addition of sodium nitrite (5-20 mM) for 72 h to mouse
    BALB/c3T3 cells resulted in the induction of transformed foci (type
    III) in a dose-dependent manner. The cells isolated from the
    NaNO2-induced transformed loci produced progressively growing
    tumours when inoculated subcutaneously into (immunodeficient) nude
    mice at an inoculation size of 1×106 cells per spot, whereas
    untreated cells did not.

         The possibility that NaNO2 reacts with cellular or medium
    components to produce carcinogenic N-nitroso compounds which in
    turn might induce cell transformation was examined and rejected.
    Thus nitrite, itself seems to have a cell transforming activity.
    Recent evidence suggest that NO2- is produced by the activated
    macrophages of mammals (Tsuda & Hasegawa, 1990).

    2.2.9  Special studies on interaction with antioxidants

         In a 4-week study, the effect of combined treatment with
    anti-oxidants, sodium ascorbate (NaAsA) and sodium nitrite on
    forestomach cell proliferation were examined in male Fischer rats.
    Groups of 5 animals (6-week old) were treated with 0.8% catechol,
    0.8% hydroquinone, 1% tert-butylhydroquinone (TBHQ), 2% gallic acid
    or 2% pyrogallol alone or in combination with 0.3% NaNO2 in
    drinking-water and/or 1% NaAsA in the diet. The thicknesses of the
    forestomach mucosa in rats treated with antioxidants and NaNO2 in
    combination were greater then those with antioxidants alone, and
    additional NaAsA treatment further enhanced the thickening of the
    mucosa (Yoshida  et al., 1994).

    2.2.10  Special studies on effects on vitamin levels

         Reported reduced vitamin A liver deposits after nitrite intake
    are probably caused by direct reaction of nitrite with carotene
    prior to absorption (WHO, 1978; Emerick, 1974).

         In two 2-week toxicity studies, chickens were fed a diet
    containing 0 or 0.4% potassium nitrite (equal to 0 or 400 mg/kg
    bw/day), and in the second study 0 or 18-60 mg/kg bw/day sodium
    nitrite. The test animals showed growth retardation, enlarged
    thyroid glands and decreased vitamin A content in the liver despite
    the vitamin A-rich diet (Sell & Roberts, 1963; Bruggemann & Tiews,

         In a number of animal species including rats, pigs, sheep and
    poultry, chronic nitrite intoxication was reported to induce
    vitamin A deficiency. The vitamin A contents in the liver were
    depleted in non-ruminants due to its degradation under acid
    conditions in the intestinal lumen (Emerick, 1974; Sell & Roberts,

         In five experiments, sodium or potassium nitrite
    (710-1830 mg/kg bw) was administered in drinking-water or in dry
    complete feed mixture to piglets or feeder pigs (5-13 animals/
    group) for 20-42 days. Administration of nitrite did not exert
    adverse effects on the metabolism of vitamin A and E (Dvorak,

    2.3  Observations in humans

    2.3.1  Methaemoglobin formation

         Nitrite is more toxic to young infants than to adults, due to
    the higher methaemoglobin formation in infants (section

         Accidental human intoxications have been reported due to the
    presence of nitrite in food. The oral lethal dose for humans was
    estimated to vary from 33 to 250 mg NO2-/kg bw, the lower doses
    applying to children and elderly people (Corré & Breimer, 1979).
    Toxic doses giving rise to induction of methaemoglobinaemia ranged
    from 1 to 8.3 mg/kg bw (Winton  et al., 1971; Simon, 1970). Several
    case reports of human intoxication from high nitrite exposure have
    recently been published (Machabert  et al., 1994; Dudley & Salomon,
    1993; Bradberry  et al., 1994; Kaplan  et al., 1990; Walley &
    Flanagan, 1987). The toxicity of nitrite can be induced both from
    inhalation (amyl nitrite) and oral intake (sodium nitrite, amyl
    nitrite). The approximate intake figures were sometimes reconstructed

    from residual nitrite in food products. Symptoms of nitrite poisoning
    and MetHb formation after ingestion ranged from 0.4 to > 200 mg/kg
    bw, expressed as nitrite ion. Symptoms of methaemoglobinaemia include
    cyanosis, euphoria, flushed face, headache, dizziness, ataxia,
    followed by dyspnoea and tachycardia, depending on the level of
    exposure to nitrite. MetHb formation in different cases varied from
    7.7 up to 79%. Patient recovered well due to therapy with methylene
    blue combined with oxygen and/or ascorbic acid and in severe cases,
    exchange transfusion (Kaplan  et al., 1990; Walley & Flanagan,
    1987). From these case reports it was deduced that cyanosis occurred
    at MetHb concentration above 10%, and other symptoms at > 20%. If no
    therapy was immediately applied, concentrations of 60-70% MetHb
    were often fatal (Kaplan  et al., 1990; Walley & Flanagan, 1987;
    Bradberry  et al., 1994). Another source of information with
    respect to nitrite toxicity in humans is the use of sodium nitrite
    as medication for vasodilation or as antidote in cyanide poisoning.
    Doses of 30-300 mg/person, equivalent to 0.5-5 mg/kg bw, did not
    cause toxic effects (NAS, 1981).

         Aside from infants under 3 months of age, several other
    categories of individuals with altered physiological status or with
    hereditary or acquired disease may also be predisposed to the
    development of nitrite- or nitrate-induced methaemoglobinaemia.
    These include pregnant women (Metcalf, 1961), individuals with
    glucose-6-phosphate dehydrogenase deficiency (Kohl, 1973), adults
    with reduced gastric acidity (including those being treated for
    peptic ulcer or individuals with chronic gastritis or pernicious
    anaemia), a rare group with a hereditary lack of NADH or
    methaemoglobin reductase activity in their red blood cells (Scott,
    1960), and probably the elderly (Spiegelhalder, in press).

         Individuals with hereditary structural abnormalities in
    haemoglobin, referred to as haemoglobin Ms, are probably also at
    increased risk from dietary nitrate or nitrite (Jaffé & Heller,
    1964, cited in NAS, 1981).

         Decreased excretion of 17-hydroxy and 17-ketosteroids occurred
    in urine of humans upon ingestion of 0.5 mg NaNO2/kg bw/day in
    cooked vegetables for 9 days. These results indicated a decreased
    production of adrenal steroid, in line with experiments reported in
    rabbits (Violante  et al., 1973). They also support a causal
    relationship between the administration of nitrite and the
    hypertrophy of the adrenal zona glomerulosa in rats (Til  et al.,
    1988, 1990; Boink  et al., in press).

    2.3.2  Relationship between nitrate and nitrite intake, the
           subsequent endogenous formation of N-nitroso-compounds and
           possible risk of (stomach) cancer in humans

         Several authors suggested that the risk for the development of
    stomach cancer is positively correlated with three factors : (i)
    nitrate level in drinking-water, (ii) urinary excretion of nitrate
    and (iii) the occurrence of atrophic gastritis (Speijers  et al.,

         During the last three decades the incidence of stomach cancer
    has been decreasing. It has been suggested that this was caused by
    factors such as the significant reduction of nitrate and nitrite
    concentrations in cured meat, and the increasing use of
    refrigerators and freezers (Hartman, 1983).

         The incidence of gastric cancer is still high in countries
    with frequent consumption of salted fish (Japan, Iceland, Chile),
    and in countries with long winters and consequently prolonged food
    preservation (Eastern Europe, Russian Federation, China).

         The daily nitrate intake seemed to be associated with the
    development of gastric cancer in a number of epidemiological and
    related studies (Weisburger & Raineri, 1975; Fraser  et al., 1980;
    NAS, 1981). Fine  et al. (1982) suggested an association between
    nitrate intake and gastric cancer mortality by combining previously
    published data on daily nitrate intake in different countries with
    gastric cancer mortality rates (r = 0.88).

         Based on the available results of epidemiological and related
    studies concerning the association between food components and the
    development of stomach cancer, two hypotheses have been proposed
    (Joossens & Geboers, 1981; Food Council, 1986): (i) the salt
    hypothesis, in which a large intake of salt is considered to be an
    important etiological factor and (ii) the nitrate/nitrite
    hypothesis as discussed in this section. Since nitrate and other
    salts are present in the diet, a combination of both hypotheses is
    likely (Correa  et al., 1975, 1983; Weisburger  et al., 1981).

         Several factors or conditions can influence the formation of
    gastric tumours. The correlation between nitrate intake and tumour
    incidence involves several factors which influence the reduction of
    nitrate to nitrite. These factors, as previously discussed, involve
    the biotransformation of nitrate, presence of thiocyanate (smokers
    versus non-smokers), iodide, age (increasing salivary nitrate and
    nitrite levels with increasing age), conditions for bacterial
    growth in the GI tract (pH of the stomach or type of indigestible

    material in the diet), and antacid medication (Armijo  et al.,
    1981 a,b; Boyland & Walker, 1974; Eisenbrand  et al., 1980;
    Forman  et al., 1985, NAS, 1981; Reed  et al., 1981; Ruddell  et al.,
    1978; Tannenbaum  et al., 1979; Tannenbaum, 1981; Ward, 1984).

         Factors influencing the formation of carcinogenic N-nitroso
    compounds are also important in correlating nitrite or nitrite
    intake with gastric tumour incidence. Factors influencing
    nitrosation of amines and amides were discussed in section
    and include the role of thiocyanate, high salt intake, pH of the
    stomach, vitamin C or other dietary components, medication
    (cimetidine and other antacid), and gastric lesions or disorders
    (Armijo  et al., 1981a,b; Forman  et al., 1985; Mirvish, 1975;
    Risch  et al., 1985; Tannenbaum, 1987).

         The majority of the studies revealed no correlation, or in
    some cases a negative correlation, between nitrate intake and
    gastric cancer. A high intake of certain vegetables, although an
    important source of nitrate, seemed to be associated with a lower
    risk of gastric cancer. Protective factors such as ascorbic acid
    simultaneously present in these foods may be involved.

         Epidemiological studies have been carried out in various
    countries on the relationship between gastric cancer and nitrate
    exposure via drinking-water. Salivary nitrite levels in volunteers
    were strongly increased after consumption of drinking-water
    containing 200 mg NO3/litre in comparison with 50 mg NO3-/litre
    (WHO guideline value). However, in studies of large populations in
    Chile, Denmark, England, France and Hungary no correlation was
    found between nitrate in drinking-water and stomach cancer. This
    still held true when the analysis was restricted to urban areas
    with nitrate levels above 50 mg NO3/litre (Hart & Walters, 1983;
    Hill  et al., 1973; Zaldivar & Wetterstrand, 1978; Juhasz  et al.,
    1980; Davies, 1980; Jensen, 1982; Vincent  et al., 1983; Beresford,
    1985; WHO, 1985; Speijers  et al., 1987).

         The originally reported positive association in females in the
    mining town of Worksop (Hill  et al., 1973) was no longer
    significant after correction for mining area and social class
    (Davies, 1980).

         No association between nitrate concentration in food alone or
    in combination with drinking-water was found in Chile and England,
    when populations from high- and low-risk areas for stomach cancer
    were compared (Armijo  et al., 1981b; Forman  et al., 1985).

         Exposure from environmental pollution sources or from food via
    natural fertilizers in Chile led to a significant association
    between nitrate load and gastric cancer (Armijo & Coulsen, 1975;
    Zaldivar, 1977; Speijers  et al., 1987).

         Studies of gastric cancer mortality in occupationally-exposed
    fertilizer workers did not show any evidence of an excess gastric
    cancer rate (Fraser  et al., 1982; Al-Dabbagh  et al., 1986).

         A study of 556 grinders occupationally exposed from 1958 to
    1976 to cutting fluids containing nitrite and amines, did not
    reveal an increased risk of cancer (Järvholm  et al., 1986).

         Individuals with an achlorhydric stomach, and persons on
    cimetidine and antacid medication do present a special risk group.
    Chronic gastritis, especially the atrophic form seems to be an
    important intrinsic factor in the genesis of stomach cancer
    (Cuello  et al., 1976; Zhang  et al.. 1984; Tannenbaum, 1987;
    Speijers  et al., 1987).

         Atrophic gastritis is a relevant factor in determining the
    gastric nitrite level, because nitrate administered to subjects
    with this type of gastritis results in a ten times higher nitrite
    concentration than that found in subjects with a normal mucosa. A
    given nitrate dose may be harmless to normal subjects, but harmful
    to a patient with atrophic gastritis, especially in the presence of
    precursors of N-nitrosamines or nitrosamides in the diet (see

         According to Ruddell  et al. (1978) iron deficient patients
    with gastric lesions and patients with pernicious anaemia (PA) are
    predisposed to stomach cancer and also have a high reduction rate
    of nitrate to nitrite. The reduction rates in PA patients were
    nearly 50-fold higher than of matched controls, as was the number
    of bacteria (Ruddell  et al., 1978; Reed  et al., 1981).

         Epidemiological case-control studies have suggested an
    association between prenatal exposure to N-nitroso compounds or
    their precursors and the appearance of tumours, including
    neuroblastomas in children, and with congenital brain defects
    (Alexandrov  et al., 1990).


         The toxic effects of nitrite are of the following three types:
    (1) the formation of methaemoglobin; (2) hypertrophy of the adrenal
    zona glomerulosa in rats; and (3) genotoxicity.

         Methaemoglobinaemia is seen particularly after acute and
    subacute exposure situations. However, it is not the sole
    determinant of the NOEL. In a 2-year oral toxicity study in rats,
    the NOEL was 6.7 mg nitrite/kg bw/day (67 mg/1 of drinking-
    water/day), expressed as nitrite ion. At the next higher dose level
    of 67 mg nitrite/kg bw/day, methaemoglobin accounted for 5% of the
    total haemoglobin; in addition, dilatation of coronary arteries and
    of the bronchi with infiltration of lymphocytes and alveolar
    hyperinflation were also seen. Methaemoglobin is particularly
    important where it exceeds 10% of total haemoglobin, leading to
    toxic effects such as cyanosis. Young infants (below the age of 3
    months) seem especially vulnerable to methaemoglobin. There is also
    evidence that fetal haemoglobin is more readily oxidized to
    methaemoglobin, and that in the neonate methaemoglobin reductase
    is less effective in the reduction of methaemoglobin to normal

         In a 90-day toxicity study in Wistar rats, the incidence and
    degree of hypertrophy of the adrenal zona glomerulosa observed at
    a dose level of 5.4 mg/kg bw/day, expressed as nitrite ion, were
    not significantly different from that among controls, whereas at
    higher dose levels the hypertrophy was both significant and

         In another 90-day toxicity study carried out by other
    investigators with a different Wistar substrain, slight hypertrophy
    of the adrenal zona glomerulosa was seen from 28 days onwards, but
    only at dose levels three times as high. The NOEL for hypertrophy
    in these studies was 5.4 mg/kg bw/day, expressed as nitrite ion.

         Nitrite both with and without nitrosatable precursors was
    found to be genotoxic in several  in vitro and  in vivo test
    systems. However, DNA repair was not affected by nitrite.

         Carcinogenicity studies with nitrite were negative, with the
    exception of those in which extremely high doses of both nitrite
    and nitrosatable precursors were administered. In addition, there
    was no evidence for an association between nitrite and nitrate
    exposure in humans and the risk of cancer. The Committee noted that
    few epidemiological studies were available in which cancer other
    than gastric cancer was investigated.

         Although it has been shown in several controlled laboratory
    studies that, when both nitrite and N-nitrosatable compounds are
    present together at high levels, N-nitroso compounds are formed
    endogenously, there are quantitative data only on those N-nitroso
    compounds which are readily formed endogenously, such as
    N-nitrosoproline, which is not carcinogenic. As there was no
    quantitative evidence of the endogenous formation of carcinogenic
    N-nitroso compounds at intake levels of nitrite and nitrosatable
    precursors achievable in the diet, a quantitative risk assessment
    of nitrite on the basis of endogenously formed N-nitroso compounds
    was not considered to be appropriate. The safety evaluation was
    therefore based on the toxicity studies on nitrite.


         As previously mentioned, the NOEL was 5.4 mg/kg bw/day
    (expressed as nitrite ion) in 90-day toxicity studies in rats in
    which hypertrophy of the adrenal zona glomerulosa was observed, and
    6.7 mg/kg bw/day (expressed as nitrite ion) in a 2-year toxicity
    study in rats in which toxic effects in the heart and lungs were
    observed. On the basis of these results and a safety factor of 100,
    the Committee allocated an ADI of 0-0.06 mg/kg bw to nitrite,
    expressed as nitrite ion. This ADI applies to all sources of
    intake. Nitrite should not be used as an additive in food for
    infants below the age of 3 months. The ADI does not apply to such


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    See Also:
       Toxicological Abbreviations
       Nitrite (JECFA Food Additives Series 50)
       NITRITE (JECFA Evaluation)