NITRATE
First draft prepared by
Laboratory for Toxicology, National Institute of Public Health
and Environmental Protection, Bilthoven, Netherlands
Explanation
Biological data
Biochemical aspects
Absorption, distribution, and excretion
Biotransformation
Endogenous synthesis of nitrate
Toxicological studies
Acute toxicity studies
Short-term toxicity studies
Long-term toxicity/carcinogenicity studies
Reproductive toxicity studies
Special studies on embryotoxicity/teratogenicity
Special studies on genotoxicity/mutagenicity
Special studies on the effects of nitrate on
the thyroid
Special studies on the effects of nitrate on gastric
epithelium
Special studies on the effects of nitrate on
behaviour
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
Relationship between nitrate intake and
genotoxic effects
Relationship between nitrate intake and
teratogenic effects
Relationship between nitrate intake and
thyroid effects
Comments
Evaluation
References
1. EXPLANATION
Nitrate was considered at the sixth, eighth and seventeenth
meetings of the Committee (Annex 1, references 6, 8, and 32). At the
sixth meeting, an ADI of 0-5 mg/kg bw, expressed as sodium nitrate,
was allocated. This ADI was based on a NOEL for sodium nitrate of
500 mg/kg bw/day derived from a long-term toxicity study in rats and a
short-term toxicity study in dogs together with a safety factor of
100. Growth depression was observed at higher dose levels. The ADI of
0-5 mg/kg bw was retained at the eighth and seventeenth meetings.
Since the previous evaluation, new toxicological and
epidemiological data have become available, which were reviewed at
the present meeting. The Committee noted that nitrate per se can
generally be considered to be of relatively low toxicity. However, it
was aware that nitrite is formed in the human body by reduction of
nitrate and that N-nitroso compounds can also be formed from nitrite
and N-nitrosatable compounds under certain conditions. Thus, the
assessment of the health risk of nitrate to humans should encompass
the toxicity of both nitrite and N-nitroso compounds, and the animal
species used for safety evaluation should be closely related to humans
with respect to the toxicokinetics of nitrate and the conversion of
nitrate to nitrite. Furthermore, in the toxicological evaluation of
nitrate, it should be considered in conjunction with nitrite and
potential endogenously formed N-nitroso compounds.
The following monograph summarizes 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
2.1.1.1 Animals
Uptake of nitrate from the upper respiratory tract occurred
within 5 minutes after intratracheal administration in mice and
rabbits (Parks et al., 1981).
After intravenous injections of 13N-labelled nitrate in mice
and rabbits, an equilibrium with extracellular fluid was obtained
within 5 minutes (Parks et al., 1981).
Intratracheal instillation of 13N-labelled nitrate in mice and
rabbits gave very similar results (Parks et al., 1981). After 20
minutes, 16% of the radioactivity from intravenously injected 13NO3
was found in the stomach and intestines of rats, 7% in the liver,
kidney and bladder, and 70% in the eviscerated carcass (Witter
et al., 1979b). Salivary glands were not examined.
Ingested nitrate is rapidly absorbed from the rat upper small
intestine with little if any absorption from the stomach and lower
intestine (terminal ileum, caecum and proximal colon). About 50% of
the radioactive label was detected in the eviscerated rat carcass
within 1 h after oral gavage of 13NO3 (Witter et al., 1979a,b;
Balish et al., 1981; Hartman, 1982; Fritsch et al., 1979; Walker,
in press).
Nitrate from blood is selectively distributed to the salivary
glands and actively secreted in saliva in humans and most laboratory
animals, but not in rats (Fritsch et al., 1985; Nighat et al.,
1981; Witter & Balish, 1979; Walker, in press). The transport of
nitrate from blood to saliva is competitively mediated by active
carriers that are shared by iodide and thiocyanate (NAS, 1981;
Edwards et al., 1954; Brown-Grant, 1961; Burgen & Emmelin, 1961).
This active transport system is lacking in rats (Cohen & Myant, 1959;
Mirvish 1983; Vitozzi, 1993; Walker, in press). However, the kinetics
of nitrate secretion in rat saliva appear to have been less well
studied than in humans, and even less is known concerning salivary
secretion in mice. This poses difficulties in interpreting the
significance for humans of toxicological studies conducted in these
species (Walker, in press). In addition to the saliva, secretion of
nitrate occurs at other sites in the GI tract leading to reduction by
the gut flora. Thus in the rat, absorbed nitrate is secreted in
gastric and intestinal secretions, including bile (Witter et al.,
1979a; Fritsch et al., 1979; Walker, in press). Unlike humans, rats
can actively secrete nitrate into the lower intestinal tract (Witter &
Balisch, 1979; Walker, in press). Absorbed nitrate may re-enter the
stomach and intestinal lumen directly via the bloodstream and via
secretions. Secretion of nitrate into the stomach may be mediated by
active carriers similar to those in the salivary glands (Edwards
et al., 1954). Nitrate in the lower intestine of rats was shown to
originate directly from the blood or intestinal secretions rather than
from the passage of gastric contents or secretions of bile and
pancreatic juice (Witter et al., 1979a). However, rats may be
exceptional in this respect as they are able to excrete iodide into
the small intestine whereas this is unlikely to occur in other animal
species (Brown-Grant, 1961; NAS, 1981). In the dog, in addition to
strong salivary secretion, large quantities of nitrate were excreted
in the bile following i.v. administration of nitrite, thus confirming
this pathway of excretion as well as oxidation of nitrite in vivo
(Walker, in press).
Nitrate levels were elevated in milk of lactating rats and cows
given high nitrate doses (Ariga et al., 1984; Nijhuis et al.,
1982). Nitrate is also frequently detected in normal cows' milk (NAS,
1981). However, the nitrate concentration in milk did not exceed the
plasma nitrate level in a beagle dog after intravenous nitrate
injection indicating that unlike salivary secretion, nitrate transport
in milk is not an active process (Green et al., 1982).
Nitrate excretion in urine generally reflects nitrate intake.
However, various authors have reported that urinary nitrate excretion
may exceed nitrate intake if the latter is low, as a consequence of
endogenous nitrate formation (see Section 2.1.3).
In conventional flora (CV) rats, approximately 55% of orally
administered 15N-labelled nitrate was excreted unaltered in urine,
and 11% was present as urinary ammonia and urea. Nitrate was not
excreted in the faeces of CV or germ-free (GF) rats thus leaving 34%
of the dose unaccounted for (Green et al., 1981a,b; Schultz et al.,
1985).
In ferrets, urinary nitrate excretion amounted to 36% of an
ingested dose. Other nitrogen compounds were not monitored (Dull &
Hotchkiss, 1984).
After absorption, nitrate rapidly equilibrates in body fluids
(Ishiwata et al., 1975a; Walker, in press). Low levels of nitrate are
normally present in body fluids and tissues of laboratory animals
(Witter & Balish, 1979; Fritsch et al., 1985). Normal plasma nitrate
levels in mongrel dogs were 6-10 mg/litre, equal to 0.1-0.15 mmol/litre
(Fritsch et al. (1985).
Fritsch et al. (1985) found that nitrate could be excreted in
the saliva and bile of dogs in concentrations similar to plasma
nitrate values.
In cattle, nitrate was absorbed from the rumen. Peak blood
nitrate levels occurred 4 h after intraruminal gavage. When the same
amount of nitrate was fed in hay, absorption was slower due to
the lower rate of uptake and nitrate levels in blood remained
substantially lower. The extent of uptake did not change (Wright &
Davison, 1964).
The elimination of nitrate from plasma varied considerably
between species. The elimination half-life of nitrate resulting from
nitrite injection was 45 h in dogs and 4 h in sheep and ponies
(Schneider & Yeary, 1975).
2.1.1.2 Humans
Nitrate is primarily absorbed from the upper part of the human
digestive tract (Bartholomew & Hill, 1984; Witter et al., 1979a).
Absorption is rapid: within 1-3 h after ingestion of nitrate in food
or drink, peak levels of nitrate were observed in serum, saliva and
urine by various investigators (Bartholomew & Hill, 1984; Ellen
et al, 1982; Spiegelhalder et al., 1976; Turek et al., 1980;
Fritsch & de Saint Blanquat, 1992).
An average 25-fold increase in plasma nitrate was found 10
minutes after ingestion of nitrate (470 mmol/kg bw). The concentration
of nitrate rose to a peak level of 1.83 mmol/litre in 40 minutes, a
value 49 times the pre-load level. Erythrocyte-nitrate followed a
similar pattern, but remained at about 2/3 of the plasma values
(Cortas & Wakid, 1991).
Absorbed nitrate is rapidly distributed to the salivary glands
and probably to other exocrine glands. After 1-3 h from ingestion, a
peak value of nitrate was observed in saliva and sweat. The increase
in the amount of nitrate secreted by the salivary glands was directly
related to the amount of nitrate ingested, although there were
marked inter-individual and diurnal variations (Walker, in press;
Spiegelhalder et al., 1976; Bartholomew & Hill, 1984; Stephany &
Schuller, 1980; Tannenbaum et al., 1976; Cortas & Wakid, 1991).
On average, 25% of oral nitrate intake was secreted in the saliva
(Stephany & Schuller, 1980; Walker, in press).
The transport of nitrate to the salivary glands is probably
mediated by active carriers. Edwards et al. (1954) reported
substrate inhibition of active iodide secretion in saliva by nitrate,
and also by thiocyanate (SCN-) and perchlorate (C104-). Thus,
SCN-, iodide and C104- would also be able to inhibit nitrate
secretion in saliva. This may be of importance for smokers who have
SCN- levels 3 to 4 times higher than non-smokers (Boyland & Walker,
1974). Forman et al. (1985) actually found lower nitrate levels in
the saliva of smokers than of non-smokers.
Salivary nitrate levels were found to be generally higher with
increasing adult age (Forman et al., 1985). However, salivary nitrate
levels depend largely on nitrate intake. Salivary concentrations
reported for adults ranged from less than 0.1 mmol/litre after low
nitrate intake (Turek et al., 1980), to over 40 mmol/litre after a
high-nitrate dose (Ellen et al., 1982). Average salivary nitrate
level reported for a group of breast- and bottle-fed infants was
0.5 mmol/litre (range 0.1-1.0 mmol/litre) (Turek et al., 1980). In
healthy volunteers administered 10 mg sodium nitrate, the cumulative
salivary nitrate excretion, over 24 h expressed as percentage of the
ingested dose, was 28% (Kortboyer et al., in press).
After i.v. administration of 13N-labelled nitrate in one
volunteer, the label was rapidly distributed in the bloodstream
throughout the body. The radioactivity accumulated almost linearly
with time in a small region of the abdomen, which was probably due to
the swallowing of salivary nitrate (Witter et al., 1979a). In a
study with healthy volunteers administered 10 mg sodium nitrate/kg bw,
the plasma nitrate half-life was approximately 6.5 h and the volume of
distribution was approximately 33 litres (Kortboyer et al., in
press).
Nitrate may be present in human milk. Levels of up to 5 mg
NO3-/kg milk were reported (Sukegawa & Matsumoto, 1975). However,
nitrate levels in milk from lactating women after a normal evening
meal did not exceed the corresponding elevated plasma nitrate levels
(Green et al., 1982).
Single oral doses of 25-170 mg potassium nitrate gave an urinary
nitrate excretion of 65-70% irrespective of the dose. Excretion was
maximal 5 h after ingestion and returned to baseline levels within
18 h. Reported urinary nitrate baseline levels in fasting subjects
were 10-20 mg/litre (Bartholomew & Hill, 1984; Tannenbaum & Green,
1981; Wagner et al., 1983a).
Small amounts of 15N-labelled ammonia and urea were found in
the urine after ingestion of 15N-labelled nitrate (Wagner et al.,
1983b).
Large single oral doses of ammonium nitrate (7-10.5 g) resulted
in an average urinary nitrate excretion of 75% within 24 h, with small
amounts of nitrite detected in only 26% of the samples. In this study,
nitrate baseline levels in urine were higher (2.4-9.3 mmol/l, equal to
149-577 mg/l), probably because the subjects were not submitted to
dietary restrictions. The mean nitrate clearance after an oral dose of
NaNO3 of 470 µmol/kg bw was 25.8 ml/minute corrected for a body area
of 1.73 m2. The urinary nitrate/creatinine ratio increased 25 to 70
times after dosing. These results indicated a predominantly tubular
excretion of nitrate (Ellen et al., 1982).
Urinary nitrate excretion in infants was reported to be 80-100%
of the average intake, but no specific data were given for exposure
levels (Turek et al., 1980).
In another study with healthy infants, the urinary excretion of
nitrate (316 mg, average 8.7 mg NO-3/day) was as high or higher
than the average (low) intake of 2-7 mg of NO-3 plus NO-2 per
day. It was concluded that excretion probably included endogenously
synthesized nitrate (Hegesh & Shiloas, 1982).
Low levels of nitrate and nitrite were detected in the faeces of
humans on a 'Western diet' with unknown nitrate content (Saul et al.,
1981). Less than 0.1% 15N-labelled nitrate was found in the faeces
of 12 volunteers ingesting 298 mg of 15N-labelled sodium nitrate.
15N-labelled ammonia and urea were also detected in small quantities
(Wagner et al., 1983b).
Incubation of nitrate with fresh human faeces under anaerobic
conditions resulted in a rapid conversion of nitrate by the faecal
microflora, suggesting that faecal excretion of nitrate may be higher
than the amount detected (Archer et al., 1981; Saul et al., 1981).
2.1.2 Biotransformation
2.1.2.1 Animals
The most important metabolite of nitrate is nitrite. However,
nitrite is converted rapidly and may not be readily detected.
Therefore, methaemoglobin formation, which is caused by nitrite (see
monograph on nitrite and section 2.1.2.3 on methaemoglobin formation
in this monograph), is often used as an indicator for nitrite
formation although it may not be a very sensitive indicator (Ward
et al., 1986). Nitrate is metabolized to nitrite and in addition it
can (via nitrite) be broken down to hydroxylamine, ammonium and
ultimately to urea (Mascher & Marth, 1993).
Part of ingested nitrate in CV rats, but not in GF rats, was
reported to be metabolized to NH4+ and urea (Green et al.,
1981a,b; Schultz et al., 1985). Bacterial reduction is an important
mechanism for nitrate conversion in mammals (Witter & Balish, 1979;
Green et al., 1981a; Schultz et al., 1985). Nitrate reductase is
present in many bacteria and other microorganisms normally present in
the GI tract (WHO, 1985).
Turek et al. (1980) observed that nitrate reduction by faecal
flora of pigs under anaerobic conditions was more rapid after a
prolonged high-nitrate diet, suggesting the possibility of bacterial
selection or induction. Wise et al. (1982) observed a several-fold
increase in nitrite production in the rat caecum when adding 5% pectin
to the diet. According to the authors, this increase could not be
attributed to overall differences in the diversity or number of
microorganisms but was likely to be due to bacterial enzyme induction.
Nitrate reductase activity has been demonstrated in various rat
tissues (WHO, 1985; Ward et al., 1986). In rats, 90% of total
mammalian tissue nitrate reductase activity was present in the liver
(Schultz et al., 1985). The same authors calculated from the urinary
nitrate excretion in CV and GF rats after intraperitoneal nitrate
injection that approximately half of the metabolized nitrate in CV
rats was metabolized by mammalian processes and the other half by
enteric bacteria.
In ferrets, 67% of a large single oral 15NO3- dose was
metabolized (Dull & Hotchkiss, 1984). The ferret may be a more
suitable experimental animal than the rat because its basal stomach
acidity and gastric morphology are more similar to those of humans.
Nitrate reduction in humans, and probably in most animal species,
takes place for the largest part in the oral cavity (saliva). It may
also occur throughout the GI tract; however, the conversion is
pH-dependent and therefore does not occur in the stomach of most
monogastric animals (Wright & Davison, 1964; Mirvish, 1975; Walker, in
press). The rumen of ruminants and the enlarged caecum and colon of
horses are especially suited for nitrate reduction due to the dense
microbial population and the relatively high pH (Wright & Davison,
1964; Sen et al., 1969; Mirvisch et al., 1975).
In rabbits and ferrets, the average gastric pH is low and
therefore considered to be similar to that of humans (Sen et al.,
1969; Dull & Hotchkiss, 1984); in cats, rats and dogs it is higher,
2.9, 4-5 and 5.4-7.4, respectively (Sen et al., 1969). In GF rats,
the pH of various parts of the GI tract is significantly higher than
in CV rats (Ward et al., 1986).
Salivary nitrate reduction is almost absent in rats (Witter
et al., 1979a; Til, 1986; Vittozzi, 1992) which is probably due to
the low salivary nitrate secretion in this species.
Although nitrate reduction in the lower part of the gut is higher
in the rat than in humans, the less efficient absorption of formed
nitrite makes the rat (and probably the mouse) different with respect
to the toxicokinetics of nitrate and thus less suitable as a model for
nitrate toxicity in humans (Vittozzi, 1992; Speijers, in press). This
conclusion is supported by comparing the NOAEL in rat studies and the
reported (sub)acute toxic effect level in humans which is 10-60 times
lower than the NOAEL in rats (Speijers, in press).
2.1.2.2 Humans
Nitrate is converted to nitrite by microorganisms in the saliva.
About 4-7% of ingested nitrate was detected as nitrite in the saliva
(Eisenbrand et al., 1980; Spiegelhalder et al., 1976; Stephany &
Schuller, 1980; Speijers et al., 1987; Brüning-Fann & Kaneene,
1993). Kortboyer et al. (in press) found in human volunteers
administered 10 mg sodium nitrate/kg bw (twice the ADI) that 8% of the
ingested nitrate was converted to nitrite. The reduction of nitrate in
the saliva accounts for 70-80% of the nitrite exposure (Bos et al.,
1985). The ratio of nitrite/nitrate in the saliva 1-2 h after intake
of various nitrate doses was remarkably constant within one individual
but differed greatly between individuals (from 0.06 to 3.6)
(Bartholomew & Hill, 1984; Ellen et al., 1982). The major site for
this reduction appears to be at the base of the tongue where a stable,
nitrate-reducing microflora is established (Walker, in press).
The concentration of salivary nitrite was directly related to
orally ingested nitrate (Stephany & Schuller, 1978; Spiegelhalder
et al., 1976; Harada et al., 1975; Ishiwata et al., 1975a,b,c).
However, Tannenbaum et al. (1976) suggested that the reduction
process may become saturated at high nitrate intakes. Oral reduction
of nitrate is the most important source of nitrite for humans and most
species (except the rat and probably the mouse) which possess an
active salivary secretory mechanism of nitrate (Stephany & Schuller,
1980; Walker, in press).
Factors that may influence the oral microbial flora are, for
example, nutritional status, infection, environmental temperature and
age. A sudden drop of temperature resulted in a dramatic fall of
salivary nitrite levels, but this may have been caused by increased
salivary flow as well as reduced microbial activity (Eisenbrand
et al., 1980). Salivary nitrite levels were generally higher in
older age groups, although considerable variation between individuals
was noted (Eisenbrand et al., 1980; Forman et al., 1985).
A low pH (1-2) in the fasting stomach is considered normal for
adults, and under these conditions bacterial nitrate reduction does
not take place because of poor bacterial growth. High gastric pH
values and sometimes correspondingly high nitrite levels were observed
in achlorhydric man, stomach cancer and gastric ulcer patients, in
patients with atrophic gastritis and patients treated with cimetidine
and antacids (Correa et al., 1975; Ruddell et al., 1976; Schlag
et al., 1982; Bartsch et al., 1984; Sen et al., 1969; Mirvish,
1975; Wright & Davison, 1964; Walker, in press). In human volunteers
administered omeprazole (pH elevating drug) followed by 10 mg
sodium nitrate/kg bw, the gastric pH was increased and the nitrite
concentration in gastric juice was approximately 6 times higher
(Kortboyer et al., in press). Studies on ileostomy patients given
a conventional or high nitrate/nitrite meal indicated that the type
of foodstuff ingested can significantly alter levels of nitrite
and nitrate in the distal ileum and is a factor in determining
nitrite/nitrate input into the proximal colon (Radcliffe et al.,
1989).
Infants younger than 3 months are highly susceptible to gastric
bacterial nitrate reduction because they have very little production
of gastric acid (Ellen & Schuller, 1983; Kross et al., 1992).
Gastrointestinal infections, which frequently occur in infants may
produce an additional increase in the reduction of nitrate to nitrite.
Contrary to the usual assumption that the normal gastric pH is
low, a high proportion of normal healthy adults (30-40%) were found to
have a fasting gastric pH >5 which was relatively stable over a
prolonged period with correspondingly high bacterial activity and high
nitrite levels (Ruddell et al., 1976; Müller et al., 1984). In one
third of 15 healthy volunteers, major variations of the fasting
gastric pH occurred occasionally, with corresponding changes in the
bacteriological parameters (Müller et al., 1984).
Schultz et al. (1985) developed a model for the fate of nitrate
in humans based on various human data tested in rats. The model
suggested that the bacteria of the large intestine were responsible
for about half of the extrarenal removal of nitrate from the body.
Ascorbic acid did not affect nitrate plasma levels nor the amount of
nitrate excreted in urine, faeces or saliva, indicating that ascorbic
acid does not interfere with nitrate metabolism (Wagner et al.,
1983b).
The half-life of nitrate in the body after ingestion was
approximately 5 h (Wagner et al., 1983b). Nitrite was not detected
in any of the body fluids studied except saliva where it appeared to
increase as nitrate levels decreased (Cortas & Wakid, 1991).
The in vivo conversion of nitrate to nitrite is complex and the
quantitative aspects are difficult to clarify because of nitrate and
nitrite endogenous synthesis, and the oxidation to nitrate of other
nitrogen-containing compounds (e.g., ammonia, hydroxylamine). In
addition, once nitrite is formed, it has a short biological half-life,
being rapidly oxidized to nitrate in the blood. Nitrate undergoes
active secretion in humans not only in the salivary duct cells but
also in the gastric pariental cells and, in passive equilibration with
other intestinal secretions, occurs at a number of other sites leading
to enterosystemic cycling of nitrate and nitrite. Because of this
complex biotransformation, the literature on nitrate provides only
qualitative or at best semi-quantitative information on nitrate
reduction, nitrite formation, and circulating methaemoglobin
levels which represent a dynamic equilibrium between oxidation of
oxyhaemoglobin by nitrite and reduction by methaemoglobin reductase.
Moreover, there are marked interspecies variations in the activity of
this enzyme in the erythrocytes (Walker, in press).
Methaemoglobin formation
As described above, nitrate is reduced to nitrite, which in
turn causes the oxidation of oxyhaemoglobin to methaemoglobin.
Methaemoglobin formation by nitrite is discussed in the monograph on
nitrite. A few studies dealing with nitrate intake and MetHb formation
are discussed here.
Normal MetHb levels in human blood range from 1%-3%. Reduced
oxygen transport was noted clinically when MetHb concentrations
reached 10% or more (Canada, 1992; Speijers, in press). The
relationship between blood nitrate and MetHb formation is not linear
at lower nitrate concentrations. A minimum amount of nitrite must
enter the bloodstream before a measurable increase in MetHb
concentration can be detected (Kross et al., 1992). Infants younger
than 3 months are particularly susceptible to nitrate poisoning
because fetal Hb is more readily oxidized to MetHb and as mentioned
before, under certain conditions the reduction of nitrate to nitrite
can be high. Pregnant women, persons with genetically controlled
deficiencies of the enzymes glucose-6-phosphate dehydrogenase or MetHb
reductase and probably the elderly are also more vulnerable to the
toxic effects of nitrate and nitrite (Corre & Breimer, 1979; Canada,
1992; Speijers, in press). Nitrates in water supplies at concentrations
above 45 mg/l as NO3- have led to numerous cases of infant
methaemoglobinaemia, particularly in infants up to 6 months of age
(Van Went & Speijers, 1989), although the role of microbial infections
may also be important (ECETOC, 1988; Van Went & Speijers, 1988;
Gangolli et al., 1994).
2.1.3 Endogenous synthesis of nitrate
2.1.3.1 Animals
When nitrate intake is low, urinary nitrate excretion usually
exceeds the intake. This was demonstrated to be the case in CV and GF
rats suggesting that nitrate was synthesized in the animals.
Furthermore, the high excretion in GF as well as in CV rats showed
that bacterial activity was not obligatory for this synthesis (Green
et al., 1981a).
The inhalation of nitrogen oxides from air could account for, at
most, 1% of the excess excreted nitrate (Green et al., 1981a).
Proof of nitrate biosynthesis was supplied by Dull & Hotchkiss
(1984) for ferrets and by Saul & Archer (1984), Wagner et al.
(1983a) and Wishnok et al. (in press) for rats. Ingestion of
15N-labelled ammonium salts was invariably followed by the urinary
excretion of small amounts of 15N-labelled nitrate. It was proposed
that ammonia is at first oxidized to hydroxylamine, catalyzed by the
generation of free radicals, which is then further oxidized to yield
nitrate (Wagner et al., 1983a). This hypothesis was confirmed by the
experimental in vivo synthesis of nitrate from hydroxylamine and the
enhanced synthesis of nitrate from ammonia by rats treated with an
endotoxin-inducing free radical formation (Saul & Archer, 1984; Wagner
et al., 1983a).
Urinary nitrate excretion increased 9 times after intraperitoneal
injection of E. coli lipopolysaccharides (Wagner et al., 1983a).
Stuehr & Marletta (1985) found that infection with Mycobacterium
bovis could increase the urinary nitrate excretion of mice from
3.6 to 164 mg/kg bw. Both studies indicated that endogenous nitrate
synthesis may increase considerably under inflammatory conditions.
2.1.3.2 Humans
Various authors reported an excess urinary nitrate excretion
(0.3-1.9 mmol/day) at low nitrate intake (<0.25 mmol/day) in humans
(Bartholomew & Hill, 1984; Green et al., 1981b; Lee et al., 1986;
Tannenbaum & Green, 1981; Wagner et al., 1983b; Gangolli et al.,
1994; Wishnok et al., in press). When large amounts of 15N-labelled
nitrate were ingested (up to 3.5 mmol), urinary excretion of unlabelled
nitrate was still considerable (0.7-1.3 mmol/day) (Green et al.,
1981b).
Ellen & Schuller (1983) calculated that up to 20% of this excess
excretion could have originated from the inhalation of NO2- and
NO3- from indoor and outdoor air and cigarette smoke. The
remaining excess urinary nitrate, up to 1 mmol/day, most probably
originates from endogenous synthesis. In vivo nitrate synthesis from
ammonia and hydroxylamine was confirmed in rats and ferrets (see
section 2.1.3.1). Although bacterial activity is not obligatory for
this synthesis in animals it may enhance nitrate biosynthesis and the
occurrence of GI infections may thus be important. Considerably
increased urinary nitrate excretion was found in infants with acute
diarrhoea (from 8.7 to 39 mg NO3- per 24 h), at low intake of
NO3- and NO2- of 2-7 mg/day (Hegesh & Shiloah, 1982).
A major pathway for endogenous nitrate production is the
conversion of arginine by macrophages to nitric oxide and citrulline,
followed by oxidation of the nitric oxide to N2O3 and the reaction
of N2O3 with water to yield nitrite. Nitrite is rapidly oxidized
to nitrate through reaction with haemoglobin. In addition to
macrophages, many cell types can form nitric oxide, generally from
arginine. The question of whether the arginine-nitrate pathway can be
associated with increased cancer risk via exposure to endogenously
formed N-nitroso compounds remains open. Nitric oxide is mutagenic
toward bacteria and human cells in culture, it causes DNA strand
breaks, deamination (probably via N2O3), oxidative damage, and can
activate cellular defense mechanisms. In virtually all cases, the
biological response is paralleled by the final nitrate levels. Thus,
while endogenously-formed nitrate itself may be of relatively minor
toxicological significance, the levels of this substance may
potentially serve as integrators for these potentially important
nitric oxide-related processes (Wishnok et al., in press; Gangolli
et al., 1994).
2.2 Toxicological studies
2.2.1 Acute toxicity studies
Oral LD50 values were 2480-6250 mg sodium nitrate/kg bw in
mice, 4860-9000 mg/kg bw in rats and 1600 mg/kg bw in rabbits. Female
rats seemed to be more sensitive than males (Mammalian Toxicity Array,
1982; Corré & Breimer, 1979).
A lethal dose of 300 mg sodium nitrate/kg bw has been reported in
pigs. The LD50 in cows following a single oral administration was
estimated to be 450 mg sodium nitrate/kg bw (Bradley et al., 1942),
whereas the LD50 was 970-1360 mg/kg bw when the total dose was
administered over a 24 h period (Crawford, 1960). The lethal dose in
cows appeared to be ten times lower than in non-ruminants (Gwatkin &
Plummer, 1946; Emerick, 1974). Acute intoxication occurred in cattle
when rations with 6% or more nitrate in dry matter were fed. Fatal
intoxications in cattle were also reported at dose levels of 1.5% in
feed (Bradley et al., 1942), whereas hay with 0.75% nitrate as dry
matter revealed no adverse effects (Geurink & Kemp, 1983).
Signs of acute nitrate intoxication varied with animal species.
Generally ruminants display methaemoglobinaemia, while monogastric
species develop severe gastritis (Brüning-Fann & Kaneene, 1993).
2.2.2 Short-term toxicity studies
2.2.2.1 Mice
In a 15-day study, male C57B1 mice received i.p. injections
of 0, 50 or 100 mg sodium nitrate/kg bw/day. Cytogenetic and
pathohistological changes of the spleen, liver and kidneys were
examined. A moderate increase in the number of macrophages was
observed in the spleen after nitrate treatment. The kidneys showed
alterations such as damaged small canals in the cortical part
reflected by dystrophic cells, cytoplasm filled with small grains and
missing or limited nuclei. In the liver, cell effects analogous to the
ones in the kidney were observed. These slight histopathoiogical
effects were reversible (Rasheva et al., 1990).
2.2.2.2 Rats
In a 4-week study, rats (10/sex/group) were fed diets containing
0, 1, 2, 3, 4 or 6% potassium nitrate or 5% sodium nitrate, equivalent
to 0, 500, 1000, 1500, 2000 or 3000 mg potassium nitrate/kg bw/day,
and 2500 mg sodium nitrate/kg bw/day. Two types of diet were used: a
cereal basal and a semi-purified diet. At 3% potassium nitrate, the
female rats had slightly elevated methaemoglobin levels and the male
rats showed increased relative kidney weights. No effects were
observed at 1% and no important differences were found between the two
types of diets (Til et al., 1985a,b).
F344 rats (10/sex/group) were fed diets containing 0, 1.25, 2.5,
5, 10 or 20% sodium nitrate, equivalent to 0, 625, 1250, 2500, 5000 or
10 000 mg/kg bw/day for 6 weeks. There was a slight or moderate
reduced weight gain in rats of the two highest dose groups. At autopsy
the abnormal colour of the blood and spleen due to methaemoglobin was
marked in rats of the two highest dose groups. From these results it
was determined that the maximum tolerated dose was 5% in the diet
(Maekawa et al., 1982).
In a 12-week study, rats were administered by gastric intubation
0 or 1/20 of the LD50 of sodium or calcium nitrate. The energy
conversion processes, such as the glycolysis and the pentose phosphate
cycle, were reported to be altered after nitrate treatment. Changes in
the glutathione-ascorbinate system of the liver and brain tissues were
also reported (Diskalenko et al., 1972a,b; cited in WHO, 1978).
In a 14-month study, rats (10/sex/group) received drinking-water
containing 0 or 4000 mg sodium nitrate/litre, equivalent to 0 or
400 mg/kg bw/day. The methaemoglobin levels in the nitrate group were
the same as in the control animals. Nitrate had a moderate effect on
plasma vitamin E level and on the incidence of chronic pneumonitis
(Chow et al., 1980).
2.2.2.3 Rabbits
In a 4-week study, rabbits (6 males/group) received 0, 200, 400
or 600 mg potassium nitrate/kg bw/day in a pulse dose via gelatin
capsules. The rabbits of all nitrate-treated groups showed
intoxication symptoms within 2 weeks, including significant weight
reduction, tachycardia, polyuria and weakness (Nighat et al., 1981).
2.2.2.4 Dogs
Three dogs (2 females, 1 male) were fed a diet containing 2%
sodium nitrate, equivalent to 500 mg/kg bw/day for 105-125 days. No
adverse effects were observed (Lehman, 1958).
2.2.2.5 Cattle
In an 8-week study, calves (12 males/group) received artificial
milk containing 18 (control group), 400, 2000, 5000 or 10 000 mg
NO3-/kg of milk. No adverse effects were observed on growth
pattern, weight gain, food conversion, biochemical blood parameters,
or morphology of the liver and kidneys (Berende et al., 1977).
2.2.3 Long-term toxicity/carcinogenicity studies
2.2.3.1 Mice
Mice (10/sex/group) received for more than 1 year diets
containing 0, 25 000 or 50 000 mg sodium nitrate/kg of feed. No
difference in tumour incidences were observed in the animals
(Greenblatt & Mirvish, 1973; Sugiyama et al., 1979, abstract only).
In an 18-month study, mice (100/group) received 0, 100 or 1000 mg
nitrate/litre of drinking-water. The concentration of urea increased
with time and nitrate dose. The mice at the high-dose group lost
weight and died prematurely. At 100 mg nitrate/l, no changes were seen
in biochemical parameters, including liver and kidney function, total
iron, ammonium, total protein and electrophoresis of the various serum
proteins and N-glycolneuraminic acid as a tumour marker (Mascher &
Marth, 1993).
2.2.3.2 Rats
In a 2-year study, rats (20/sex/group) were fed a diet containing
0, 0.1, 1, 5 or 10% sodium nitrate. At the 5% dose level a slight
growth inhibition was observed, whereas inanition was noticed at the
10% dose level. Complete histopathological examination, including
tumour incidences, was performed. No abnormalities or increased tumour
incidence were found. The NOEL in this study was 1%, equivalent to
500 mg sodium nitrate/kg bw/day, or 370 mg/kg bw/day expressed as
nitrate ion (Lehman, 1958; Annex 1, references 6 & 33).
In a carcinogenicity study, rats (15/sex/group) received 0 or 5%
sodium nitrate/l of drinking-water for 84 weeks and were killed 20
weeks later. Histopathological examination did not reveal any increase
in tumour incidence. (Lijinsky et al., 1973).
In a 2-year carcinogenicity study, F344 rats (50/sex/group)
received diets containing 0, 2.5 or 5.0% sodium nitrate, equivalent to
0, 1250 or 2500 mg sodium nitrate/kg bw/day, or 0, 910, or 1820 mg/kg
bw/day expressed as nitrate ion. No carcinogenic effects were detected.
This strain of rats is known to have a high incidence of mononuclear
leukemia which was higher in controls than in the experimental
groups (Maekawa et al., 1982).
2.2.4 Reproductive toxicity studies
2.2.4.1 Guinea-pigs
Groups of female guinea-pigs received during 143-204 days 0
(4 animals), 300 (6), 2500 (3), 10 000 (3) or 30 000 (3) mg potassium
nitrate/l in drinking-water, equal to 0, 12, 102, 507 or 1130 mg
potassium nitrate/kg bw/day. The mating behaviour was highly impaired
at 30 000 mg/l and the number of pregnant animals was seriously
reduced. The fertility of the animals of other nitrate groups was not
reduced since pregnancy occurred in all groups. Weight gain and food
and water intake were normal at all concentrations. No macroscopic or
microscopic alterations were observed in the reproductive organs
(Sleight & Atallah, 1968).
2.2.4.2 Rabbits
Rabbits were given 0, 250 or 500 mg nitrate/l during 22 weeks. No
detrimental effects on reproductive performances were found after
successive gestations. Measures of reproductive performance included
fertility, litter size or weight at birth and at weaning, plasma
retinol and progesterone concentration and Hb level. However, a
decrease in retinol concentration in the liver of progeny of exposed
rabbits (themselves exposed for 5 weeks to nitrate in drinking-water)
was observed. Hb level was slightly decreased in dams given 500 mg/l
(Kammerer, 1993; Kammerer & Siliart, 1993).
2.2.4.3 Sheep
Sheep (5/group) were fed a diet containing 0.3, 0.6 or 1.2%
NO3- in drinking-water from day 21-49 of pregnancy till parturition
(for a total of 41-74 days). These doses were high enough to induce
severe methaemoglobinaemia, however, no changes in abortion rates were
observed (Davison et al., 1965).
2.2.4.4 Cattle
In a 7-month study, 15 heifers were fed a diet containing 445 or
665 mg NO3-/kg of feed from 2 months of pregnancy until birth. No
treatment-related changes in pregnancy were observed, although the
dose levels selected led to 20-50% methaemoglobinaemia. Macroscopical
examinations revealed no abnormalities in the newborn animals (Winter
& Hokanson, 1964).
The effect of high-nitrate oat hay on late-gestating (46 days
prior to parturition) crossbred beef cows (8 cows/group) and their
subsequent calves was studied over a 92-day period. The results of the
study suggested that up to 1.4% KNO3 in the diet may not cause
abortions in cows during late gestation when fed under controlled
conditions. However, this level of nitrate appeared to cause loss of
cow body weight (Hixon et al., 1992).
2.2.5 Special studies on embryotoxicity/teratogenicity
No data available
2.2.6 Special studies on genotoxicity/mutagenicity
Nitrate did not induce mutagenic effects in bacterial tests with
Salmonella typhimurium. When tested under aerobic and anerobic
conditions in Escherichia coli, mutagenicity was only found under
anaerobic conditions. The mutations, however, were probably due to the
reduction of nitrate to nitrite under the test conditions (Konetzka,
1974).
In an in vitro chromosome aberration test with hamster cells,
sodium nitrate revealed mutagenic effects, whereas with potassium
nitrate negative results were obtained. Sodium chloride - in contrast
to potassium chloride - was also positive at high concentrations in
the same test system (Ishidate et al., 1984). It is likely that
interactions may have taken place between the chromosomes and elevated
concentrations of the sodium ions which subsequently led to chromosome
aberrations (Ashby, 1981).
In acute experiments, mice were treated intragastrically with
doses of 79, 236, 707 or 2120 mg sodium nitrate/kg bw. An increase in
chromosome aberrations was found at only one dose (707 mg/kg bw) and
the number of micronuclei was enhanced at 79 and 236 mg/kg bw. At
doses of 707 mg/kg bw and higher, cytotoxicity occurred in the bone
marrow as shown by a concomitant depression of the bone marrow. In
contrast, acute treatment of rats with doses up to 2120 mg/kg bw did
not show chromosome abnormalities in the bone marrow. However, rats
subacutely treated with the same doses of sodium nitrate, showed a
significantly enhanced number of chromosome aberrations in bone
marrow. According to the authors, it cannot be excluded that formation
of N-nitroso compounds was responsible for the bone marrow damage
(Luca et al., 1985).
Oral administration of 500 mg sodium nitrate/kg bw to pregnant
Syrian hamsters on days 11 or 12 of gestation did not lead to an
increase in gene mutations, chromosome abnormalities, micronuclei or
morphological transformation in cells cultured from the hamster
embryos (Inui et al., 1979). However, Rasheva et al. (1990) found
induction of chromosome aberrations in male C57B1 mice after 5 and 15
day treatment with 50 or 100 mg sodium nitrate/kg bw.
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 was found with dietary nitrate, nitrite and 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).
2.2.7 Special studies on the effects of nitrate on the thyroid
In rats, doses of 40-4000 mg NO3-/l in drinking-water for 100
days had no effect on the serum iodine level or protein-bound iodine.
Minor changes were reported in 131I uptake by the thyroid, thyroid
weight and the histology of the thyroid. These effects were seen at all
dose levels, but there was no dose-response relation (Höring, 1985;
Höring et al., 1988; Seffner, 1985).
Potassium nitrate was administered to 56-day old pigs at a
dietary concentration of 3% for 2 days or 6 weeks (equivalent to
730 mg/kg bw/day expressed as nitrate ion). Levels of MetHb, serum
T4 ,T3, nitrate and somatomedin were determined. Sufficient iodine
uptake by mothers prevented a decrease in T4 levels after
administration of KNO3 for 2 days. After 6 weeks of treatment,
however, the decrease in T4 level could not be prevented by
supplementing the diet with 0.5 mg iodine/kg bw. A decrease in serum
somatomedin activity due to nitrate administration was also observed
which correlated with a decreased body-weight gain in pigs (Jahreis
et al., 1987).
2.2.8 Special studies on the effects of nitrate on gastric epithelium
In a 19-month study, Wistar rats were fed twice a week a dose of
0.1 of the LD50 of nitrate. Ultrastructural examination showed that
sodium nitrate alone or in combination with saphrol caused atypical
changes in the gastric epithelium (Ptashekas, 1990).
2.2.9 Special studies on the effects of nitrate on behaviour
The development of sensoro-motor functions and adult learning
behaviour was studied in rats exposed to nitrate. Pregnant and
lactating dams (50/group) and their offspring were supplied with
drinking-water containing 0, 1.12 or 2.24 mmol KNO3/litre (equal to
0, 113 or 226 mg/l). Postnatal maturation of reflexes, that of sensory
and somatic parameters and motor activity, the acquisition of one-way
avoidance and rewarded discriminative learning behaviour in adulthood
were examined. Reflexes (righting, cliff avoidance) and hearing
startle reaction maturated earlier in the nitrate treated groups. Open
field motor activity was higher at days 5, 7, and 10 after birth, but
hypoactivity ensued after day 20. A marked learning deficit was
observed both in punished and in rewarded learning tests. The results
indicated a nitrate-induced deviation in behavioural development, and
an impairment in learning behaviour, particularly of the discriminative
type (Markel et al., 1989).
2.3 Observations in humans
The toxicity of nitrate in humans, as well as in animals, depends
on the conversion of nitrate to nitrite. For this reason infants and
patients with hypo- or achlorhydria and/or stomach lesions are to be
considered as special risk groups. These patients might also be more
susceptible to the toxicity of nitrate (Speijers et al., 1987;
Brüning-Fann & Kaneene, 1993; Speijers, in press).
Human lethal doses of 4-50 g NO3- (equivalent to 67-833 mg
NO3-/kg bw) have been reported. Toxic doses - with methaemoglobin
formation as a criterion for toxicity - ranged from 2 to 5 g (Corré &
Breimer, 1979) and 6 to 9 g of NO3- (Fassett, 1973). These values
are equivalent to 33-83 and 100-150 mg NO3-/kg bw, respectively.
Fassett (1973) reported a rapidly occurring severe gastroenteritis
with abdominal pain, blood in the urine and faeces as symptoms of
acute nitrate intoxication. Repeated doses gave rise to dyspepsia,
mental depression, headache and weakness. Farre et al. (1982)
reported on nine cases of mild methaemoglobinaemia which appeared as
an outbreak in a group of 50 infants. The cause of intoxication was an
excessive concentration of nitrate (76 mg/l) in well water.
Eighty cases of acute nitrate poisoning were reported from 1973
to 1989 by Gao & Guo (1991). The patients came to the emergency
department of the hospital. Most patients were in shock with moderate
respiratory distress, pallor or cyanosis in the mouth and extremities
and abnormalities in mental status. RBC was normal lot all patients.
WBC was temporarily higher in 16 cases. In 2 cases, ASAT and BUN
levels were elevated. It was assumed that each patient ingested more
than 2 g nitrate.
The data on nitrate toxicity in humans originate partly from
relatively old publications, some of which do not provide details on
age or gastric conditions. The low values of these lethal and toxic
doses are difficult to interpret. Contradicting these values are
reports of absence of toxic symptoms in 12 volunteers receiving
intravenously 9.5 g of sodium nitrate in 1 h, while in 2 of 12 other
persons administered 7-10.5 g of ammonium nitrate orally in one dose,
vomiting and diarrhoea occurred (Ellen et al., 1982). The lethal
dose of nitrate in adults is probably around 20 g NO3-, equivalent
to 330 mg NO3-/kg bw (Leu et al., 1986; Ellen, 1986). In infants
under the age of 3 months the conversion of nitrate to nitrite and
methaemoglobin formation is high as discussed previously in section
2.1.2.2 and in the monograph on nitrite. Gastrointestinal disturbances
play a crucial role, the reduction of nitrate to nitrite in the
stomach being enhanced by bacterial growth at the high pH in the
stomach of these infants. Toxic effects are therefore induced at a
much lower dose of nitrate than in adults. According to Corré &
Breimer (1979) assuming an 80% reduction of nitrate to nitrite in
these young infants, the toxic dose was calculated to vary from
1.5-2.7 mg NO3-/kg bw, using 10% formation of methaemoglobin as
toxicity criterion (Winton et al., 1971). In the same report a
lethal dose for infants of 43.2 mg NO3-/kg bw was calculated based
on haemoglobin/methaemoglobin transfer stoichiometry (WHO, 1985).
However, in reported cases of infant methaemoglobinaemia, the amounts
of nitrate ingested were higher: 37.1-108.6 mg/kg bw, with an average
of 56.7 mg/kg bw.
Acute intoxications have been reported due to drinking of well
water containing high nitrate levels (WHO, 1978; NAS, 1981). Of all
reported cases of infantile methaemoglobinaemia, 97.7% occurred in
areas with a nitrate content in drinking-water of more than
44.3-88.6 mg NO3-/1 (WHO, 1985). In the Netherlands, these
intoxications have occurred sporadically in the last two decades.
However, the evaluation of cases of infantile methaemoglobinaemia in
relation to nitrate intake is difficult because of the frequent
occurrence of simultaneous bacterial contamination of drinking-water
and of bacterial infections in infants which may influence the
reduction of nitrate to nitrite as well as the endogenous synthesis of
nitrate. Hegesh & Shiloah (1982), for example, found a significantly
increased nitrate blood content, paralleling an increased
methaemoglobin content, in infants with acute diarrhoea, whereas the
intake of nitrate and nitrite by these infants was low (2-7 mg/day)
(See also section 2.1.3).
In healthy infants 11 days to 11 months of age, oral treatment
for several days with 50 or 100 mg NO3-/kg bw increased
methaemoglobin levels (5.3-7.5%) but no cyanosis was seen. In 6-7 week
old infants just recovering from previous methaemoglobinaemia after
administration of 100 mg NO3-/kg bw, cyanosis and increased
methaemoglobin concentrations (up to 11%) were found. Details of
individual infants age and days of treatment were not given (Cornblath
& Hartmann, 1948).
2.3.1 Relationship between nitrate intake, the subsequent endogenous
formation of N-nitroso-compounds and possible risk of (stomach)
cancer in humans
Several authors have suggested that the risk for the development
of stomach cancer is positively correlated with three factors: 1) the
nitrate level of the drinking-water, 2) the urinary excretion of
nitrate and 3) the occurrence of atrophic gastritis.
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).
Siddiqi et al. (1992) presented analytical data on aliphatic
amines and nitrate from the most commonly used fresh and sun-dried
vegetables, red chillies and salted tea from a high risk area for
oesophagal and gastric cancer in Kashmir. Exposure estimates for the
adult population showed high nitrate intake (237 mg/day) and
exceptionally high exposure to N-nitrosatable compounds such as
methylamine (1200 µ/day), ethylamine (14 320 µg/day), diethylamine
(400 µg/day), dimethylamine (150-280 µg/day), pyrrolidine (517 µg/day)
and methylbenzylamine (40 µg/day).
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; Dutt et al., 1987). 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
research 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 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; Weisburger et al., 1981).
Several factors or conditions can influence the formation of
gastric tumours (Speijers et al., 1987; Moller, in press). The
correlation between nitrate intake and tumour incidence involves
several factors which influence the reduction of nitrate to nitrite.
These factors, discussed in detail in section 2.1.2, involve the
biotransformation of nitrate, the 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., 1981a,b; Boyland &
Walker, 1974; Eisenbrand et al., 1980; Forman et al., 1985;
NAS, 1981; Reed et al., 1981b; Ruddell et al., 1978; Tannenbaum
et al., 1979; Ward, 1984).
Factors influencing the formation of carcinogenic N-nitroso
compounds are also important in correlating nitrate or nitrite intake
with gastric tumour incidence. Factors influencing nitrosation of
amines and amides were discussed in the Monograph on nitrite 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, 1985; Risch et al., 1985).
These factors are discussed in many epidemiological and related
studies concerning nitrate or nitrite intake and the occurrence of
gastric tumours (Speijers et al., 1987; Brüning-Fann & Kaneene,
1993).
Some studies support the claim that there is evidence for a
correlation between gastric cancer and nitrate (nitrite) intake. On
the other hand there are also studies which do not support an
association between high nitrate levels and increased incidence of
gastric cancer. The majority of the studies were inconclusive or in
some cases revealed a negative correlation between nitrate intake and
gastric cancer (Speijers et al., 1987; Forman, 1987; Forman et al.,
1988; Hansson et al., 1994; Bruning-Fann & Kaneene, 1993;
Moller et al., 1994; Gangolli et al., 1994; Speijers et al., in
press). Epidemiological studies (on cancer) in general are hindered by
a variety of factors such as the multiplicity of gastric cancer
etiological factors and the time lag between exposure and the
development of cancer (Brüning-Fann & Kaneene, 1993; Gangolli et al.,
1994).
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 (Buiatti et al., 1989, 1990;
Boeing et al., 1991; Gangolli et al., 1994; Moller, in press).
Epidemiological studies have been carried out in several
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-/l in comparison with 50 mg NO3-/l (WHO guideline
value). However, in studies of large populations in Chile, Denmark,
England, France, Hungary and the USA 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 the 50 mg/l (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; Rademacher
et al., 1992).
The originally reported positive association in females in the
mining town of Worksop (Hill et al., 1973) was no longer significant
after correction for mining area and social class (Davies, 1980).
No association between nitrate concentration in food alone or in
combination with drinking-water was found in Chile and England, when
populations from high- and low-risk areas for stomach cancer were
compared (Armijo et al., 1981b; Forman et al., 1985).
Exposure from environmental pollution sources or from food via
natural fertilizers in Chile led to a significant association between
nitrate load and gastric cancer (Armijo & Coulson, 1975; Zaldivar,
1977; Speijers et al., 1987; Moller, in press).
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;
Rafnsson and Gunnardottir, 1990; Hagmar et al., 1992; Fandrem et al.,
1993; Speijers et al., 1987). In addition no increase in lung or
prostate cancer was found in nitrate fertilizer workers (Hagmar
et al., 1991; Rafnsson & Gunnarsdottir, 1990).
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; Rufu et al., 1984).
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 (see
section 2.1.2). 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 also Monograph on nitrite - endogenous nitrosation).
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).
2.3.2 Relationship between nitrate intake and genotoxic effects
In an attempt to apply genetic biomarker analysis to improve the
basis for risk assessment with respect to nitrate contamination of
drinking-water, a study evaluated peripheral lymphocyte chromosomal
damage in human populations exposed to low-, medium- and high-nitrate
levels in private water wells in the Netherlands. It was shown that
nitrate contamination of drinking-water caused dose-dependent
increases in nitrate body loads as monitored by 24-h urinary nitrate
excretion in female volunteers, but this appeared not to be associated
with peripheral lymphocytes sister chromatid exchange frequencies
(Kleinjans et al., 1991).
2.3.3 Relationship between nitrate intake and teratogenic effects
The relationship between maternal exposure to nitrates in
drinking-water and risk of delivering an infant with CNS malformation
was examined in a case-control study in New Brunswick, Canada.
Exposure to nitrate levels of 26 mg/l from private well water sources
was associated with a moderate but not statistically significant
increase in risk for CNS malformation If the source of drinking-water
was a municipal distribution system or a private spring, an increase
in nitrate exposure was associated with a decrease in risk of
delivering a CNS-malformed infant. However, these estimates of effects
were not statistically significant (Arbuckle et al., 1988).
To investigate the relationship between community drinking-water
quality and spontaneous abortion, trace element levels in the
drinking-water of 286 women having a spontaneous abortion through 27
weeks of gestation with that of 1391 women having live births were
compared. After adjustment for potential confounders, a decrease in
the frequency of spontaneous abortion was associated with high levels
of alkalinity and sulfate, and any detectable level of nitrate
(Aschengrau et al., 1989).
The relationship between community drinking-water quality and the
occurrence of late adverse pregnancy outcomes was investigated by
conducting a case-control study among women who delivered infants
during August 1977 through March 1980 at Brigham and Women's Hospital
in Massachusetts. The water quality indices were compared among 1039
congenital anomaly cases, 77 stillbirth cases, 55 neonatal death
cases, and 1177 controls. There was no relationship between nitrate
levels and late adverse pregnancy outcomes or neonatal death cases
(Aschengrau et al., 1993).
2.3.4 Relationship between nitrate intake and thyroid effects
Van Maanen et al. (1994) studied the effect of nitrate
contamination of drinking-water on volume and function of the thyroid
in human populations exposed to different nitrate ion levels in their
drinking-water. No iodine deficiency was observed in any of the
nitrate exposure group. A dose-dependent difference in the volume of
the thyroid was observed between low-and medium- versus high-nitrate
exposure groups, showing development of hypertrophy at nitrate levels
exceeding 50 mg/l. An inverse relationship was established between the
volume of the thyroid and serum thyroid stimulating hormone (TSH)
levels. These effects are supported by similar findings in rats and
pigs (see 2.2.7).
3. COMMENTS
As the toxicity of nitrate results from its conversion to nitrite
and the possible endogenous formation of N-nitroso compounds, and the
toxicokinetics and biotransformation of nitrate in the rat are
different from those in humans, rats are less suitable than rabbits,
dogs and pigs for use in assessing the toxicity of nitrate in humans.
However, the toxicological data are too limited to allow a safety
evaluation on the basis of the results of studies on these species.
For these reasons both the toxicity studies on nitrate in laboratory
animals and those on nitrite in combination with data on the
conversion of nitrate to nitrite were considered by the Committee.
The possible endogenous formation of N-nitroso compounds from
nitrite and N-nitrosatable compounds as precursors has already been
discussed in the Monograph on nitrite. No evidence of an association
between nitrate exposure and the risk of cancer was found in either
the toxicological or epidemiological studies, and nitrate was not
genotoxic.
In two long-term toxicity studies in rats, one old and one
recent, doses of 370 and 1820 mg/kg bw/day, expressed as nitrate ion,
respectively failed to produce any effects. However, the second of
these was solely a carcinogenicity study, in which the highest dose
level of 1820 mg nitrate ion/kg bw/day could not be considered as a
NOEL because complete histopathological examinations were not
performed.
The experimental design of a recent study in rats on possible
behavioural effects of nitrate was considered to be inappropriate for
safety evaluation purposes.
A short-term toxicity experiment in pigs indicated that a daily
dose level of 3% potassium nitrate, equivalent to 730 mg/kg bw/day
expressed as nitrate ion, inhibited the functioning of the thyroid.
This finding was supported by an epidemiological cohort study in
which enlargement of the thyroid and decreased levels of serum
thyroid stimulating hormone were seen at high nitrate levels in
drinking-water.
4. EVALUATION
In the light of the overall information on the toxicity of
nitrate, the NOEL of 370 mg nitrate ion/kg bw/day was considered to be
the most appropriate for safety evaluation.
If the proportion of nitrate converted to nitrite in humans is
taken as 5% (mol/mol) for normally responding individuals and 20% for
those showing a high level of conversion and the NOEL for nitrite
(6 mg/kg bw/day expressed as nitrite ion) is used, the "transposed"
NOELs for nitrate, expressed as nitrate ion, would be 160 and 40 mg/kg
bw/day, respectively. As these figures are derived in part from human
pharmacokinetic data, the use of a safety factor of less than 100
is justified. If the data on individuals showing a high level of
conversion are used, a safety factor of 10 would be justified because
intraindividual differences have already been taken into account.
Since uncertainties still exist with respect to the possible
endogenous formation of N-nitroso compounds after nitrate exposure,
the most appropriate approach at present is to derive an ADI based on
the most sensitive toxicity criteria for nitrite in rats and the
toxicokinetics of nitrate in humans, in addition to deriving an ADI
directly from toxicity studies with nitrate.
On the basis of the NOEL of 370 mg of nitrate ion/kg bw/day in
the long-term study in rats and a safety factor of 100, an ADI of
0-5 mg/kg bw, expressed as sodium nitrate, or 0-3.7 mg/kg bw,
expressed as nitrate ion, could be allocated. On the basis of the
"transposed" NOEL for nitrate of 160 mg/kg bw/day for normally
responding individuals in the human population (5% rate of conversion)
and a safety factor of 50, an ADI of 0-3.2 mg/kg bw, expressed as
nitrate ion, could be allocated. Both ways of deriving an ADI for
nitrate thus give similar figures. The Committee therefore retained
the previous ADI of 0-3.7 mg/kg bw, expressed as nitrate ion. This ADI
is expressed to two significant figures because rounding up was not
considered to be justified on the basis of the value of 3.2 mg/kg bw
derived from conversion of nitrate to nitrite.
Because nitrate may be converted to nitrite in significant
amounts and infants below the age of 3 months are more vulnerable to
the toxicity of nitrite than adults, the ADI does not apply to such
infants.
In deriving an ADI for nitrate the Committee took a cautious
position. It was aware that vegetables are an important potential
source of intake of nitrate. However, in view of the well-known
benefits of vegetables and the lack of data on the possible effects of
vegetable matrices on the bio-availability of nitrate, the Committee
considered it to be inappropriate to compare exposure to nitrate from
vegetables directly with the ADI and hence to derive limits for
nitrate in vegetables directly from it.
Submission of the results of studies in humans exposed to nitrate
from different sources (vegetables and drinking-water), including the
toxicokinetics and relevant toxicodynamic parameters such as thyroid
function and adrenal cortex function, is desirable. The results should
be analyzed by means of physiologically based pharmacokinetic (PBPK)
models.
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