(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
Absorption, distribution, and excretion
Formation of N-nitroso compounds
Effects on enzymes and other biochemical parameters
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
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. BIOLOGICAL DATA
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).
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).
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).
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 &
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
220.127.116.11 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,
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.
18.104.22.168 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
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 &
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.
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).
22.214.171.124 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
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).
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).
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
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).
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
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
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).
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
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).
126.96.36.199 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 &
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
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 &
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 &
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 (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).
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
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, 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
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
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.
188.8.131.52 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 184.108.40.206).
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 220.127.116.11
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
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
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