TR-1242-60B
THE DRINKING WATER
CRITERIA DOCUMENT ON NITRATE/NITRITE
Prepared Under
Program No. 1524
for
EPA Contract 68-C8-003303-3279
ERG Subcontract No. LSI-8700
ERG Work Assignment No. 2-10
Life Systems, Inc. Work Assignment No.
for
Criteria and Standards Division
Office of Drinking Water
Environnental Protection Agency
December 21, 1990

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. DISCLAIMER
This document has been reviewed 1n accordance with U.S. Environmental
Protection Agency policy and approved for publication. Mention of trade
names or commercial products does not constitute endorsement or recommenda-
tion for use.
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FOREWORD
Section 1412 (b)(3)(A) of the Safe Drinking Water Act, as amended in 1986,
requires the Administrator of the Environmental Protection Agency to publish
maximum contaminant level goals (MCLGs) and promulgate National Primary
Drinking Water Regulations for each contaminant, which, in the judgment of the
Administrator, may have an adverse effect on public health and which is known
or anticipated to occur in public water systems. The MCLG is non-enforceable
and is set at a level at which no known or anticipated adverse health effects
in humans occur and which allows for an adequate margin of safety. Factors
considered in setting the MCLG include health effects data and sources of
exposure other than drinking water.
This document provides the health effects basis to be considered in
establishing the MCLGs for nitrate and nitrite. To achieve this objective,
data on pharmacokinetics, human exposure, acute and chronic toxicity to
animals and humans, epidemiology and mechanisms of toxicity were evaluated.
Specific emphasis is placed on literature data providing dose-response
information. Thus, while the literature search and evaluation performed in
support of this document was comprehensive, only the reports considered most
pertinent in the derivation of the MCLGs are cited in the document. The
comprehensive literature data base in support of this document includes
information published up to May, 1990; however, more recent data may have been
added during the review process.
When adequate health effects data exist, Health Advisory values for less than
lifetime exposures (One-day, Ten-day and Longer-term, approximately 10% of an
individual's lifetime) are included in this document. These values are not
used in setting the MCLGs, but serve as informal guidance to municipalities
and other organizations when emergency spills or contamination situations
occur.
Michael B. Cook
Director
Office of Drinking Water
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TABLE OF CONTENTS
LIST OF FIGURES		vi
LIST OF TABLES 		vii
I.	SUMMARY		1-1
II.	PHYSICAL AND CHEMICAL PROPERTIES		II-1
A.	Physical and Chemical Properties 		II-1
B.	Manufacture and Uses		II-3
C.	Summary		11-4
III.	TOXICOKINETICS		III-l
A.	Absorption		III-l
B.	Tissue Distribution 		III-3
C.	Metabolism		III-7
1.	Reduction in Nitrate in Saliva		Ill-7
2.	Reduction in Nitrate in the Stomach		Ill-8
3.	Oxidation of Hemoglobin by Nitrite 		Ill-10
4.	Reduction of Methemoglobin in Blood 		Ill-11
5.	Reactions of Nitrite in the Stomach 		Ill-13
D.	Excretion 		111-15
E.	Bioaccumulation and Retention 		Ill-18
F.	Summary		111-18
IV.	HUMAN EXPOSURE		IV-1
V.	HEALTH EFFECTS IN ANIMALS 		V-l
A.	Short-Term		V-l
1.	Lethality		V-l
2.	Methemoglobinemia 		V-3
3.	Effects of Blood Pressure 		V-5
4.	Histopathological Effects 		V-5
5.	Effects on Thyroid Function 		V-7
B.	Longer-Term		V-8
1.	Methemoglobinemia/Hematology 		V-8
2.	Histopathological Effects 		V-12
3.	Other Effects		V-16
C.	Reproductive/Teratogenicity Effects 		V-17
D.	Mutagenicity		V-29
E.	Carcinogenicity		V-36
F.	Summary		V-49
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VI.	HEALTH EFFECTS IN HUMANS		VI-1
A.	Methemoglobinemia		VI-1
1.	Methemoglobinemia in Infants 		VI-2
2.	Methemoglobinemia in Older Children and Adults ....	VI-10
B.	Teratogenicity and Reproductive Effects 		VI-11
C.	Other Noncancer Effects 		VI-13
D.	Mutagenicity		VI-14
E.	Cancer		VI-14
1.	Epidemiological Studies		VI-14
2.	In Vivo Formation of Nitrosamines		VI-24
F.	Summary		VI-25
VII.	MECHANISMS OF TOXICITY		VII-1
A.	Cardiovascular Effect		VII-1
B.	Effect on Hemoglobin		VII-1
C.	Diuretic Effects 		VII-2
D.	Effects Due to N-Nitrosation		VII-2
E.	Summary		VII-4
VIII.	QUANTIFICATION OF TOXICOLOGICAL EFFECTS 		VIII-1
A.	Noncancer Effects 		VIII-7
1.	Nitrate 		VIII-7
2.	Nitrite			VIII-12
a.	Extrapolation from Nitrite Data in Animals ....	VIII-12
b.	Extrapolation from Nitrate Toxicity Data in
Humans 		VIII-13
B.	Cancer 		VI1I-17
1.	Weight-of-Evidence Evaluation 		VIII-17
2.	Quantification of Cancer Risk 		VIII-17
C.	Existing Guidelines and Standards 		VIII-18
D.	Sensitive Subpopulations 		VIII-18
E.	Summary	VIII-20
IX.	REFERENCES		IX-1
APPENDIX 1 EPA Response to Review Comments Provided by the
Science Advisory Board 		Al-1
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LIST OF FIGURES
FIGURE mi
III-l Reduction of Methemoglobin 	 III-3
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LIST OF TABLES
TABLE	PAGE
II-l Properties of Nitrate and Nitrite		II-2
V-l Oral LD50 of Nitrate and Nitrite 		V-2
VIII-1 Summary of Studies on Methemoglobinemia in Infants ....	VIII-9
VIII-2 Summary of Studies on the Noncancer Effects of Nitrite
in Animals	VHI-13
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I. SUMMARY
Nitrate (N03") is a relatively stable component of the natural
environment. It exists mainly in combination with alkali metal cations such
as Na+, K+ or Ca+2, all of which are readily soluble in water. The main use
for nitrate is as a fertilizer, but is also used in explosives and
glassmaking, and as a food preservative.
Nitrite (N02") is produced by chemical or bacterial reduction of nitrate.
It is used mainly as a food preservative. Nitrite is less stable than nitrate
and many undergo a variety of reactions. With respect to health effects, the
most important of these reactions are; 1) oxidation of other substances
(e.g., Fe+2 in hemoglobin to Fe+3 in methemoglobin) , and 2) reaction with
secondary and tertiary amines to form nitrosamines.
Toxicokinetics
The primary route of nitrate and nitrite absorption is through the
gastrointestinal tract. Nitrate is absorbed by active transport through the
wall of the upper small intestine. Nitrite is absorbed by diffusion across
the gastric mucosa and also through the wall of the intestinal tract. In both
cases, absorption following ingestion is essentially complete (i.e., 100%).
Nitrate and nitrite can also be absorbed into the blood through the lungs.
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Nitrate is rapidly disseminated throughout the body in the blood and is
actively secreted in saliva, gastric juices, and urine. Nitrite reacts so
rapidly with hemoglobin that little is transported to the tissues. Nitrate
and nitrite do not bioaccumulate. However, they are constantly present in the
body as a result of de novo synthesis as well as ingestion.
Nitrate is reduced to nitrite by the action of oral and gastrointestinal
bacteria. In healthy adults and older children, approximately 5% of an
ingested dose of nitrate is converted to nitrite. This occurs mainly in the
mouth, due to the action of oral bacteria on nitrate secreted in the saliva.
In infants, nitrate may also be reduced to nitrite in the stomach. This is
because the infant stomach may have a pH sufficiently high to permit bacterial
growth. No quantitative data are available on the amount of nitrate that is
reduced in the alimentary tract (mouth, stomach) of susceptible infants, but
it is generally believed to be considerably higher than in adults. Nitrite
that is absorbed into the blood reacts with hemoglobin, oxidizing it to
methemoglobin. Nitrite in the stomach may react with amines and other
nitrogenous substrates present in food or in the epithelium to form N-nitroso
compounds.
Nitrate is excreted readily through the kidneys. When excess nitrate is
not present to the diet, both rats and humans excrete significantly more
nitrate than they ingest, revealing the importance of endogenous formation of
nitrate in vivo. Nitrite that is ingested or formed in vivo is so reactive
that none remains to be excreted.
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Health Effects in Animals
The toxic effects of nitrate are closely related to its conversion to
nitrite by bacteria in the alimentary tract. Thus, the toxicity of nitrate
depends not only on dose, but also on the level and type of bacteria present.
Consequently, dose-response relationships are quite variable between different
animal species. For example, ruminants are highly sensitive, while dogs have
low sensitivity. Because of this species variability, animal data should be
extrapolated to humans with caution.
The principal health effect from exposure to nitrate or nitrite is
methemoglobinemia. This results when nitrite oxidizes the Fe*2 form of iron
in hemoglobin to the Fe° state. In rats, acute oral LDJ0 values for NaNO,
range from 1,000 to 2,000 mg/kg. These are close to LD50 values for NaCl,
suggesting that acute lethality from NaN03 is caused mainly by nonspecific
electrolyte imbalance. Chronic exposure to doses up to 69 mg nitrate-
nitrogen/kg/day did not produce methemoglobinemia in rats, but doses of
50 mg/kg/day or higher have been noted to produce mild histological changes in
liver or spleen. High doses may also interfere with thyroid function.
Nitrite is substantially more toxic to rodents than nitrate. Acute oral
LD30 values range from 40 to 120 mg/kg (20-40 mg nitrite-nitrogen/kg/day),
while doses of 20 mg nitrite-nitrogen/kg/day can produce methemoglobinemia and
other related histological effects. High doses of nitrite act on vascular
smooth muscle to produce vasodilation and hypotension, and may also cause
changes in coronary blood vessels.
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Numerous studies on reproductive function, teratogenicity and fetal
development in several animal species have not revealed evidence of adverse
effects from nitrate or nitrite except at relatively high dose levels. Such
effects are likely to be secondary to maternal or fetal methemoglobinemia and
related toxicity. Exposure of males to high doses may result in cytogenetic
abnormalities in spermatocytes and morphological abnormalities in sperm, but
this did not result in any apparent effect on reproduction or offspring.
Ingestion of 20-100 mg nitrite-nitrogen/kg/day by dams during lactation can
cause iron deficiency in milk and moderate to severe anemia and other post-
natal effects in sucklings.
Most investigations on the mutagenicity of nitrate and nitrite have been
negative. However, many studies have reported that both nitrate and nitrite
lead to a variety of chromosomal aberrations, both in cultured cells irj vitro
and in animals exposed in vivo.
The carcinogenic potential of nitrate has not been extensively studied in
animals, but several investigations have not revealed any evidence of
increased tumor incidence. The carcinogenic potential of nitrite has been
much more thoroughly studied. One early study was interpreted as providing
evidence of increased tumors of the lymphoreticular system in rats, but
re-evaluation of the histological slides from this study led to the conclusion
that the lesions were not neoplastic. Numerous other studies of nitrite
carcinogenicity in animals have also been negative. However, when nitrite is
given to animals along with high levels of nitrosable amines, clear increases
in tumor incidence are often observed in a variety of tissues. This
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tumorigenic response is probably a result of the formation of carcinogenic
nitrosamines by reaction of nitrite with the nitrosable substrates in the
stomach.
Health Effects in Humans
The principal adverse health effect associated with exposure of humans to
nitrate is methemoglobinemia in infants (age 0-3 months). This group is much
more susceptible than older children and adults because the pH of the infant
stomach can often be sufficiently high (above pH 4) to permit growth of
bacteria that reduce nitrate to nitrite. Infants also ingest more water per
unit body weight (about 0.16 L/kg/day) than older children (about
0.1 L/kg/day) or adults (about 0.029 L/kg/day).
Low levels of methemoglobin occur in normal individuals, with typical
values usually ranging from 0.5% to 2.0%. However, due to the large excess
capacity of blood to carry oxygen, levels of methemoglobin up to around 10%
are not associated with any significant clinical signs. Concentrations above
10% may cause a bluish-color to skin and lips (cyanosis), while values above
25% lead to weakness, rapid pulse and tachypnea. Death may occur if
methemoglobin values exceed 50%-60%. Many studies have been performed on the
levels of nitrate in water that lead to clinically significant
methemoglobinemia in infants. These studies have shown that most cases are
associated with exposure to water containing 20 mg nitrate-nitrogen/L or
higher, and that no significant effects occur if water contains 10 mg nitrate-
nitrogen/L or less. In adults, methemoglobinemia has only been reported
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following accidental ingestion of large quantities of sodium nitrite (usually
mistaken for table salt).
Only two studies are available on possible developmental or reproductive
effects of nitrate in humans. One study suggested that there might be an
increased risk of birth defects associated with ingestion of water containing
5-15 mg nitrate-nitrogen/L, but there were a number of other factors that
could also have been responsible for the observation. No significant relation
between nitrate levels in water and birth defects was detected in the second
study.
Many epidemiological studies have been performed to determine if
ingestion of nitrate or nitrite are associated with increased cancer risk.
Some of these studies have noted increased cancer risk (especially
gastrointestinal cancer) in populations ingesting above average levels of
nitrate or nitrite in water or food. However, these studies are not adequate
to show that nitrate/nitrite caused the increased risk, since reliable dose-
response data are usually lacking, and there are many other risk factors for
gastrointestinal cancer that might have been responsible.
Quantification of Toxicological Effects
Clinically significant signs of methemoglobinemia are not observed in
infants exposed to drinking water containing 10 mg nitrate-nitrogen/L or
lower. This value is supported by many epidemiological studies and case
reports from the United States and elsewhere. This concentration corresponds
to an average daily intake of 1.6 mg nitrate-nitrogen/kg/day by an infant, and
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this is taken to be the Reference Dose (RfD) for nitrate for an infant. This
value is also protective for older children and adults, since infants are the
most sensitive subpopulation.
Data are not available to identify the NOAEL for nitrite in infants.
Based on the assumption that infants convert at least 10* of ingested nitrate
to nitrite, the NOAEL is estimated to be at least 1 mg nitrite-nitrogen/L.
Based on this, the RfD for nitrite is 0.16 mg nitrite-nitrogen/kg/day for an
infant. This value is also protective for older children and adults, since
they are somewhat less susceptible to nitrite-induced methemoglobinemia than
infants.
The USEPA is currently evaluating the evidence that ingestion of nitrate
or nitrite may be associated with increased risk of cancer. Cancer
weight-of-evidence categories have not yet been assigned.
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II. PHYSICAL AND CHEMICAL PROPERTIES
A. Physical and Chemical Properties
Some important physical and chemical properties of nitrate and nitrite
are summarized in Table II-l. Nitrate ion (N03~) is the physicochemically
stable form of combined nitrogen for oxygenated aqueous systems. The nitrate
salts of all common metals are quite soluble in water. Nitrate has little
tendency to form coordination complexes with metal ions in dilute aqueous
solution. In dilute aqueous solutions, nitrate is chemically unreactive, and
nearly all of the transformations involving nitrate in natural waters are
mediated biochemically (NAS 1978).
Nitrite ion (N02") contains nitrogen in an intermediate and relatively
unstable oxidation state. Both chemical and biological processes can result
in the further reduction of nitrite to more stable compounds or in oxidation
back to nitrate. Enzymatic processes in certain bacterial, fungal and plant
systems can reduce nitrate and both oxidize and reduce nitrite as part of the
natural nitrogen cycle (NAS 1978).
Nitrite ion is the conjugate base of a weak acid, nitrous acid (HN02).
In most natural waters, the acid is dissociated into ions, but in acidic
environments, a significant fraction can exist as the undissociated species.
Of the many reactions involving nitrous acid, the reaction with amino
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Table II-l Properties of Nitrate and Nitrite
Nitrate
Nitrite
Formula
Usual state
Acid
Salts
Reactivity
NOj"
Ion in aqueous solution
at neutral pH
Conjugate base of strong
acid HN03, pKa - -1.3
Nearly all soluble in
water, dissociated at
neutral pH
Unreactive
N02*
Ion in aqueous solution at
neutral pH
Conjugate base of weak acid
HN02, pKa - 3.4
Forms salts with
Li
Na
K
Ca
Sr
Ba
Ag
Reactive
Oxidizes antioxidants
Oxidizes Fe^ of hemoglobin
to Fe^
Oxidizes primary amines
Nitrosates secondary amines,
tertiary amines, ureas,
carbamates, guanidines
Adapted from NAS 1978, 1981
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substrates to form N-nitroso compounds is of special concern because many
N-nitroso compounds are carcinogenic (NAS 1978).
Nitrosation in aqueous nitrous acid at pH<5 proceeds by reaction of the
unprotonated amino substrate with an electrophilic nitrosating agent such as
dinitrogen trioxide (N203) or the hydrated nitrosonium ion (H20+-N0). Basic
secondary amines react most rapidly at pH 3.4, but these reactions are
generally slower than those for amides, ureas and carbamates (NAS 1981).
Nitrosation of secondary amines by aqueous nitrous acid is accelerated by
nucleophilic anions such as thiocyanate and iodide. Comparable nitrosation
reactions of amides, ureas, guanidines and carbamates are not usually
accelerated by these catalysts (NAS 1981).
Nitrosation of secondary amines, amides, ureas and guanidines by aqueous
nitrous acid can be inhibited by compounds that reduce nitrous acid to nitric
oxide. These inhibitors include ascorbic acid (vitamin C), a-tocopherol
(vitamin E) and several naturally occurring polyphenolic antioxidants (NAS
1981) .
B. Manufacture and Uses
The major use of nitrate is in inorganic fertilizers. A production
summary of the principal inorganic nitrogen fertilizers for August 1981, as
reported by the U.S. Bureau of the Census, in short tons (2,000 lbs) is as
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follows: ammonia - 1,510,000; ammonium nitrate - 617,000; nitric acid -
657,000 CUSDC 1981). Other uses include the production of explosives and the
production of purified potassium nitrate for glassmaking.
Sodium nitrite is produced for use as a preservative in cured meats.
Nearly four billion kilograms of cured meat products were processed with added
nitrite in the United States during 1979. Nitrate is also added to a variety
of products to serve as a source of nitrite by bacterial reduction of nitrate
to nitrite. This serves to inhibit further bacterial growth (NAS 1981).
C. Summary
Nitrate (N03~) is a relatively stable component of the natural
environment. It exists mainly in combination with alkali metal cations such
as Na+, K* or Ca+2, all of which are readily soluble in water. The main use
for nitrate is as a fertilizer, but is also used in explosives and
glassmaking, and as a food preservative.
Nitrite (N02") is produced by chemical or bacterial reduction of nitrate.
It is used mainly as a food preservative. Nitrite is less stable than nitrate
and many undergo a variety of reactions. With respect to health effects, the
most important of these reactions are: 1) oxidation of other substances
(e.g., Fe+Z in hemoglobin to Fe+3 in methemoglobin), and 2) reaction with
secondary and tertiary amines to form potentially carcinogenic nitrosamines.
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III. TOXICOKINETICS
A. Absorption
Witter et al. (1979b) administered oral radioactive nitrate ion (13N03~)
(dose not reported) to two male volunteers and followed the radioactive label
with a computer-linked Auger camera. One subject received nitrate orally
about an hour after a large meal. The half-time for the disappearance of the
label from the stomach was about 30 minutes. The radioactivity in the pylorus
remained almost constant, suggesting that nitrate is not rapidly absorbed from
the stomach but exits into the small intestine. The second subject was given
nitrate about ten hours after eating. The half-time for the disappearance of
the label from the stomach of the second subject was less than ten minutes.
A similar study was performed (doses not reported) with Sprague-Dawley
rats of undisclosed sex, some of which were previously ligated at the pyloric
valve (Witter et al. 1979a). The group with pyloric ligation retained nearly
all of the radioactivity in the stomach 40 to 50 minutes after administration.
Only 21% of the radioactivity was retained in the rats without pyloric
ligation, suggesting that nitrate leaves the rat stomach predominantly through
the pyloric valve and is not absorbed from the stomach. In the rats which
were not ligated, essentially all of the radioactivity passed into the upper
small intestine, where most was absorbed. Similar studies using 13N02~
resulted in some gastric absorption, but most of the radioactivity seemed to
exit the stomach via passage into the duodenum. The exit of nitrite from the
stomach was slower, and less radioactivity appeared to be absorbed from the
small intestine than with nitrate.
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Witter and Balish (1979) studied oral absorption of nitrate and nitrite
ions using a spectrophotometry assay to differentiate the ions from their
metabolites. Sprague-Dawley rats (60- to 90-days*old, sex not reported) were
exposed separately to both nitrate and nitrite in drinking water at 1 mg/mL
(160 mg/kg/day; equivalent to 36.2 and 48 mg/kg/day of nitrate- and nitrite-
nitrogen, respectively) for an unreported period. Various parts of the
intestinal tract were removed and analyzed for nitrate and nitrite ion
content. Rats fed nitrate had both nitrate and nitrite in the stomach, but
only nitrate in the small intestine. Rats fed nitrite had both ions in the
stomach and small intestine. Control rats had trace amounts of only nitrate
in their stomachs. These results are in agreement with the above results
(Witter et al. 1979a, 1979b), suggesting that nitrate is absorbed from the
upper small intestine and nitrite from the stomach. However, when a large
dose of nitrite is administered, some passes into the intestine.
Friedman et al. (1972) performed a colorimetric assay of nitrite
absorption in male Swiss ICR/Ha mice weighing between 20 and 25 grams. Mice
were given food and water ^d lib while 150 fig of sodium nitrite (6.8 mg/kg;
1.4 mg/kg as nitrogen) was administered to each mouse by gavage. Groups of
13-18 mice were sacrificed within 1 minute and at 10, 20 and 30 minutes after
sodium nitrite administration, their stomachs were removed and assayed. At
ten minutes after oral administration, 85% of the administered sodium nitrite
had disappeared from the mouse stomach. Ligation of the gastroduodenal
junction had no effect on nitrite absorption. Other experiments were
performed using isolated mouse stomachs where absorption of nitrate was
prevented. After a 30-minute incubation, there was 63% loss of nitrite, of
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which 40% had been converted to nitrate. The authors concluded that the major
pathway for disappearance of available gastric nitrite is absorption directly
from the stomach into the bloodstream. The rate of disappearance of nitrite
from the mouse stomach fits second-order kinetics.
Parks et al. (1981) studied the absorption of 13N03" and 13N02" after
intratracheal instillation of 10-100 ng/kg (0.00001 to 0.0001 mg/kg) in groups
of 10-12 Balb/C mice. In this study 100% of the label was absorbed within
10 minutes.
No information on dermal absorption of nitrate or nitrite was found in
the literature.
B. Tissue Distribution
Witter et al. (1979b) gavaged nine rats (sex and strain not reported)
with l3N03" (dose not reported) and 45-60 minutes later organs were removed and
the radioactivity measured. The largest fraction, 47.3%, was found in the
carcass after removal of abdominal organs. This was more than could be
accounted for as blood-bound activity. To study the fate of blood nitrate,
the sane authors injected 13N03" intravenously into nine rats of which one was
ligated at the pyloric junction and three at the ileocecal junction. Except
for the stomach, which contained 5.6%-6.8% of the radioactivity, no marked
accumulation was seen in any organ at 30 minutes and the radioactivity was
mainly in the eviscerated carcass (74.2%-78.8%). Thirty minutes after
injection, the intestinal tract from the duodenum to the large intestine
contained essentially the same amount of radioactivity even if the pylorus was
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ligated. This indicated that the label of 13N03~ could enter the intestines
from the blood, either as part of the biliary or pancreatic secretions or by
direct secretion from the bloodstream. Data from rats ligated at the
ileocecal junction suggested secretion directly into the lower intestines from
the blood.
Witter et al. (1979b) also injected 13N03" intravenously into a human
volunteer and followed the radioactivity with an Auger camera. There was
rapid distribution of the label. The radioactivity in the heart region
reached a maximum of about 3% of the administered dose at 2 minutes, as it
passed through the heart for the first time. The activity then fell rapidly
in the next 2 minutes as the radioactive label became evenly distributed in
the blood. The radioactivity accumulated almost linearly with time in a fist-
sized region of the abdomen, probably due to the swallowing of salivary 13N03~.
The remainder was fairly evenly distributed throughout the body, with less
than 5% measured in the urine or salivary glands within 40 minutes.
Parks et al. (1981) studied the distribution of 13N03~ and 13N02~ after
intratracheal instillation in Balb/C mice and intravenous injection in mice
and New Zealand White rabbits. Mice were sacrificed from 5 to 30 minutes
after administration of the labeled ions and organs were excised and weighed.
Tracer concentrations were determined by gamma-ray counting. Both tracers
achieved transient steady-state very rapidly for each route of administration.
The distribution data were relatively constant within 5 minutes. The specific
activities did not differ significantly between organs or routes of
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administration. The authors concluded that either the ions distribute equally
into the same body space or they chemically transform into each other or into
similar products.
The rabbits injected intravenously were studied with the Auger scintilla-
tion camera and by blood analysis. A rapid homogeneous distribution of the
radioactivity throughout the rabbit was observed. Steady-state between the
intravascular and extravascular compartments was reached within 5 minutes
after injection of either radiochemical. Only 2%-3% of the label appeared in
the bladder during the first 30 to 45 minutes after injection (Parks et al.
1981).
Portions of each supernatant from the blood distribution experiments in
mice and rabbits were analyzed by high-pressure liquid chromatography (HPLC).
The chromatographic results obtained from mouse plasma taken 10 minutes after
intratracheal instillation of labeled nitrite revealed that 70% had been
converted to nitrate, 27% remained nitrite and about 3% was in nonionic
compounds. The product distribution in rabbit plasma 10 minutes after
intravenous injection of labeled nitrite was 51% nitrate, 46% nitrite and 3%
in nonanionic compounds. Ten minutes after administering labeled nitrate to
both species, the label found in blood remained nitrate. Any nitrite formed
by the bacterial reduction of nitrate was not detectable (Parks et al. 1981).
Edwards et al. (1954) studied the secretion of ionic compounds in normal
people and patients undergoing treatment with radio-iodine (niI"). Numbers of
subjects and doses employed were not reported. Anions were given as oral
doses of the potassium salt and thiocyanate was detected by chemical assays.
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They concluded that chere is antagonism between iodide, thiocyanate,
perchlorate, and nitrate for salivary secretion, thus suggesting a common
transport mechanism for these ions. Nitrate was less effective than
thiocyanate or perchlorate in depressing salivary secretion of iodide, and
less effective than iodide or perchlorate in depressing salivary secretion of
thiocyanate.
Spiegelhalder et al. (1976) used a colorimetric assay to study secretion
of nitrate in saliva of 11 volunteers after ingestion of vegetables
and vegetable juices. Nitrate concentration was determined at intervals of 30
or 60 minutes for 7 hours. The increase in the amount of nitrate secreted by
the salivary glands was found to depend directly on the amount of nitrate
ingested above a threshold dose of 54 mg. About 20%-25% of ingested nitrate
is secreted. The concentration rises 30 minutes after nitrate ingestion and
peaks from 1 to 3 hours later depending on the food or juice consumed.
Swallowed salivary nitrate recirculates through the blood to the saliva again,
causing one or more additional smaller peaks of secretion.
Secretion of nitrate by the gastric mucosa was studied by Bloomfield
et al. (1962) in pylorus-ligated rats of an unreported strain. Doses of
sodium nitrate ranging from 60 to 200 mg/kg (equivalent to 9.9-33 mg/kg as
nitrogen) were injected intraperitoneally into fasted rats. As the plasma
concentration increased, the gastric juice concentration also increased. The
gastric juice to plasma ratio dropped from a value of 20 when the nitrate
concentration in plasma was 1 mM to unity at 4.5 mM. This suggests that an
active transport mechanism is involved and it can be saturated. When nitrate
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and niI" were injected simultaneously, there was less radioactivity in the
gastric juice than when 131I~ was injected alone. This suggests that nitrate
and iodide have a common concentrating mechanism in the rat stomach.
C. Metabolism
1. Reduction of Nitrate in Saliva
Nitrate can be reduced to nitrite and then to ammonia and other products
by bacteria. The reduction of nitrate in saliva by human oral bacteria was
studied by Goaz and Biswell (1961). Saliva was obtained by paraffin
stimulation, collected in test tubes immersed in crushed ice, and used in less
than an hour. Nitrate and nitrite concentrations were determined
colorimetrically. When whole saliva containing 28 mg/L nitrate (6.4 mg
nitrate-nitrogen/L) was incubated at 37*C, nitrate concentration decreased
rapidly and was not detectable after 75 minutes. Nitrite accumulated
transiently, increasing to 5 mg/L (1.5 mg nitrite-nitrogen/L) at 45 minutes
and then decreasing to zero by 105 minutes of incubation. Saliva depleted of
its endogenous nitrate and nitrite by prior incubation will rapidly reduce
added nitrate. Further experiments determined that nitrate reduction to
nitrite in human saliva occurs under either aerobic or anaerobic conditions,
but the presence of oxygen completely inhibits the reduction of nitrite.
Maximum activity occurred between pH 6 and 6.4, with no reduction of nitrate
below pH 4 or above pH 9. Between pH 8.2 and 8.6 nitrate is reduced to
nitrite but nitrite reduction is inhibited.
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Eisenbrand et al. (1980) studied salivary nitrate reduction using 11
volunteers who bad ingested varying amounts of nitrate in red beet juice or
spinach. Using a colorimetric assay, they showed a direct correlation between
the amounts of nitrite produced in saliva and the amounts of nitrate ingested.
About 20% of the secreted nitrate was converted to nitrite. The same authors
determined the mean and range of nitrite concentrations in saliva of different
age groups in Germany. There was a steady increase from a mean of 0.6 mg/L at
1 week to 6 months of age, up to a mean of 6.8 mg/L in adults.
2. Reduction of Nitrate in the Stomach
Franklin and Skoryna (1971) studied the survival of microorganisms in the
gastric lumen of fasting human subjects. The subjects were 13 hospitalized
adults having sterilized nasogastric tubes in place. Sterile samples of
gastric fluid were taken hourly and cultured. It was concluded that the
survival of bacteria in the gastric lumen depends on the pH of the gastric
juice. At pH 4 or higher, all samples showed growth of microorganisms and the
percent of samples with few or no organisms increased with decreasing pH.
When a rapid drop in pH occurred in a patient previously measured at pH
greater than 4, the clearing of viable organisms occurred within 1 hour in
most cases. The pH of a normal adult human stomach is less than 3.
Lactobacilli in breast-fed infants do not reduce nitrate to nitrite and
maintain a pH low enough to discourage other bacteria from colonizing the
infant's stomach. Marriott and Davidson (1923) determined the normal acidity
of infants' stomachs (under 1-year-old) using four-color pH indicators to
assay samples of gastric contents. Samples were withdrawn through a rubber
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cacheter 2 hours after feeding different diets. Three mL of gastric contents
were dialyzed against 3 mL of sterile physiologic saline for 15 minutes. The
dialysate was assayed for pH and compared with the standard buffer solution.
The gastric pH of 23 breast-fed normal infants averaged 3.75. When these
infants were fed undiluted cow's milk, which has a high buffering capacity,
their average gastric pH rose to 5.3. This is high enough to allow heavy
bacterial growth. In another group of infants accustomed to cow's milk, the
average pH following a meal of undiluted whole cow's milk was 4.75, indicating
an adaptation to this type of food. A group of 16 infants aged 3 weeks to
9 months who were hospital patients with acute and chronic infections were
studied similarly. While fed breast milk, their average gastric pH was 4.74.
After a feeding of undiluted whole milk the pH was 5.35, slightly higher than
in the normal infants discussed above. These authors reported that at pH 5
marked inhibition of the growth of members of the colon, dysentery and typhoid
group of bacilli occurs. At pH 4 complete inhibition or death of these
microorganisms takes place.
Bartholomew et al. (1980) studied microbial growth and nitrite formation
in the stomachs of an unspecified number of patients with pernicious anemia
and 31 ulcer patients being treated with a drug to reduce gastric acidity.
Details of their assays were not given. They found a relation between the pH
of the gastric contents and the numbers of bacteria present. Also a close
relationship was found between the pH and the percentage of nitrate reduced to
nitrite in the gastric contents. They concluded that when the pH is above 5
there is a relatively rich flora and a relatively high nitrate reductase
activity.
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3. Oxidation of Hemoglobin by Nitrite
The main fate of nitrite in blood is reaction with hemoglobin to yield
methemoglobin. Burrows (1979) characterized the action of nitrite in blood on
hemoglobin using mature ewes of mixed breeding. A 3% solution of sodium
nitrite in sterile physiological saline was administered intravenously in
doses of 6.6-100 mg/kg (1.3-20.3 mg/kg as nitrogen) to groups of 4 ewes. The
nitrite ion oxidized divalent ferrous hemoglobin to trivalent ferric
methemoglobin, which is incapable of transporting molecular oxygen. The
administration of various doses of sodium nitrite resulted in the gradual
formation of up to 80% methemoglobin. Doses of 6.6, 22, 35, or 50 mg/kg
resulted in maximum methemoglobin levels of 13% at 15 minutes, 43% at
45 minutes, 63% at 60 minutes, or 80% just before death after 60 minutes,
respectively.
The normal methemoglobin level in human blood is variously reported
between 0.5 and 2.5% of the total hemoglobin. However, Skrivan (1971)
determined methemoglobin levels of 100 pregnant women at 2-week intervals.
There was a consistent elevation of methemoglobin concentration from the
14th week of pregnancy through delivery, with the average level rising from 2%
at the 12th week to 10.5% at the 30th week. This was followed by a decline to
5.5% at the 38th week. All levels returned to normal after delivery. Women
around the 30th week of pregnancy would be unusually sensitive to nitrite
induction of clinical methemoglobinemia.
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4. Reduction of Methemoglobin in Blood
The major system responsible for methemoglobin reduction in mammalian red
cells is methemoglobin reductase or diaphorase. As indicated in Figure III-l,
this intracellular enzyme requires NADH as a cofactor. Normal human and most
mammalian erythrocytes possess a second reductive system that requires NADPH
as a cofactor. In most species, the system appears to be dormant, and it is
activated only in the presence of exogenously added electron carriers such as
methylene blue. The enzyme reduces methylene blue which in turn
nonenzymatically reduces methemoglobin (Burrows 1979).
There are large species differences in the rate of reaction of nitrite
with hemoglobin to form methemoglobin. Formation is paralleled by similar
differences in the rate of reduction by diaphorase to form functional
hemoglobin (Smith and Beutler 1966). This is important in determining how
sensitive a species is to the vasodilator effects of nitrite and resulting
cardiovascular collapse. Species which rapidly remove nitrite from the plasma
by reaction with hemoglobin (e.g., ruminants) do not show cardiovascular
effects. Species which remove nitrite by this mechanism very slowly (e.g.,
horses) die of cardiovascular collapse before forming enough methemoglobin to
appear cyanotic. Humans fall between these species in removal rate and
exhibit both kinds of symptoms concurrently. Different individuals respond
with varying sensitivity.
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NAD
NADH
Glycolysis
He tHb-reductase
MetHb-reductase
red
ox
MetHb
Hb
Methylene blue^^
Methylene blue
M.B. reductase
M.B. reductase
ox
NADPH
NADP

G6PD, pentose phosphate shunt
The spontaneous (NADH) and the dormant (NADPH) methemoglobin reductase
systems. Methemoglobin (MetHb) reductase Is active in intact red cells in the
presence of substrates that can generate NADH. The NADPH system requires
intact red cells, glucose or its metabolic equivalent, a functioning pentose
phosphate shunt, and methylene blue (M.B.). M.B. reductase reduces M.B.,
which in turn nonenzymatically reduces MetHb. Adapted from Smith (1980).
Figure III-I Reduction of Methemoglobin
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Gruener et al. (1973) determined activity of methemoglobin reductase in
six pregnant rats (strain not given), 10 rat fetuses, 49 pregnant women, and
cord blood of 69 human newborns. The activity of rat fetus methemoglobin
reductase was nearly ten times higher than that of adult rats or that found in
human cord blood. Human adult blood had 1.5 times the activity of the human
cord blood. These findings indicate the possibility that the human fetus
might have a weaker defense mechanism against intrauterine exposure to
nitrites than that detected in rats. In rats, nitrite has been shown to cross
the placenta (Shuval and Gruener 1972).
5. Reactions of Nitrite in the Stomach
Sander et al. (1968) demonstrated the reaction of nitrite with secondary
amines to form nitrosamines in the stomachs of Sprague-Dawley rats.
Twenty-four rats were fed a standard diet (amount not reported) with 0.01%
diphenylamine and 0.15% sodium nitrite and killed after 30, 60, 120, or 180
minutes. Stomach contents were extracted with dichloromethane for
two-dimensional chromatography to detect diphenylnitrosamine. The amount
found reached a maximum of 31% of theoretical at 120 minutes, presented as
37 Mg/g stomach contents. vitro studies indicated that the reaction is
optimum at pH 1 to 3. This corresponds to the conditions found in the stomach
of humans and various experimental animals. Weakly basic amines were
converted into nitroso compounds up to a thousand times more rapidly than
strongly basic amines, which could not be shown to nitrosate in the rat
stomach.
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Mirvish ec al. (1973) determined the kinetics of nitrosation of the amino
acids proline, hydroxyproline and sarcosine by sodium nitrite in vitro using
perchloric acid to control the reaction pH. The authors found a pH optimum of
2.5. Thiocyanate increased the rate of sarcosine nitrosation 10- to 400-fold,
especially at low pH.
Ohshima and Bartsch (1981) determined endogenous formation of N-nitroso-
proline in a male volunteer who ingested 0-325 mg nitrate (0-73.5 mg as
nitrogen) in red beet juice (0-4.64 mg/kg) followed 30 minutes later by
0-500 mg proline (0-7.14 mg/kg). Urine was monitored for N-nitroso compounds.
The amount of nitrosoproline excreted was found to be proportional to the
proline dose and increased exponentially with the nitrate dose ingested.
Neither nitrate nor proline taken alone led to a detectable increase. The
man's normal excretion of nitrosoproline was <3 Jig/L. Administration of
nitrate followed by proline increased this to a maximum of 17-30 fig/L at the
highest doses tested. This was a good test system for demonstrating
nitrosation in the human stomach because proline is an amino acid found in
most protein foods, and nitrosoproline is neither volatile nor carcinogenic in
animals.
Fine et al. (1982) compared the in vivo data of Ohshima and Bartsch
(1981) with the ljj vitro data of Mirvish (1975). The in vivo data fall within
theoretical limits predicted if 0.5% to 1.5% of ingested nitrate is reduced to
nitrite per hour in human saliva. This study indicates that the extent of
in vivo formation of other more harmful N-nitroso compounds in the human
stomach can be predicted from lj\ vitro kinetic studies.
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Archer et al. (1975) demonstrated the ability of ascorbic acid to block
nitrosation of morpholine in vitro by rapidly reducing nitrite to nitric oxide
or nitrogen. The reaction kinetics are dependent on the redox potential of
the medium. Under anaerobic conditions, one mole of ascorbate will reduce two
moles of nitrite. In the presence of bubbling air equimolar ascorbate is
required. These experiments were conducted at pH 3, 3.5 and 4 maintained by
perchloric acid.
Similar ia vitro studies were done by Kanm et al. (1977) showing the
ability of a-tocopherol (vitamin E) to remove nitrite ion from simulated
gastric fluid. The rate of reaction was shown to be optimum at pH 2 to 3 and
negligible at pH 5. The a-tocopherol reacted more readily than ascorbate to
destroy nitrite ion at pH values below U. Appropriate controls were used by
these authors.
Ohshima and Bartsch (1981) confirmed the inhibitory value of ascorbic
acid and o-tocopherol in the human volunteer protocol presented above. Intake
of 1 gram of ascorbic acid simultaneously with 325 mg nitrate (73.5 mg as
nitrogen) and 250 mg proline reduced the nitrosoproline concentration in urine
to the control level, whereas 500 mg of a-tocopherol inhibited nitrosation
about 50%.
D. Excretion
Keith et al. (1930) studied excretion of nitrates in four young men.
After ingestion of 10 grams of ammonium nitrate, a measurable amount of
nitrate could not be detected in the feces. Seventy-nine percent of the
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nitrate ingested was excreted promptly, and a total of 88% was recovered
within 3-4 days. Nitrate was assayed with a colorimetric test using
diphenylbenzidine.
Tannenbaum et al. (1980) recorded urinary excretion of nitrate by
individuals on a low-nitrate soy diet for 84 days. The average daily
excretion was about six times the nitrate intake. The data suggest a fairly
constant introduction of extra-dietary nitrate into the system. Based on
these data and the authors' previous demonstration of nitrite in ileostomy
contents in the absence of nitrate, the authors conclude that the most likely
explanation is that gut microorganisms oxidize reduced forms of nitrogen, such
as ammonium compounds, to nitrite in the intestinal tract. This nitrite is
absorbed and is then converted to nitrate by the process of methemoglobin
formation and subsequent reduction.
Green et al. (1981) studied nitrate excretion in germ-free or
conventional male Sprague-Dawley rats weighing 150 grams. The basal diet was
supplemented with 0%, 0.001% or 0.005% Na15N0j (0, 0.5 or 2.5 mg/kg/day;
equivalent to 0, 0.08 or 0.4 mg/kg/day as nitrogen). Urine and feces were
collected during 24-hour intervals. Nitrate content was determined by
reducing it to nitrite and assaying by diazotization and coupling with a
Griess reagent. Isotope levels were determined by conversion to nitrobenzene
and analysis by gas chromatography-mass spectrometry. Rats not supplemented
with sodium nitrate excreted ten times more nitrate in the urine than they
ingested. There was no difference between germ-free and conventional rats.
This indicates that endogenous nitrate synthesis is not due to intestinal
flora. Rats supplemented with nitrate at the lower dose (0.5 mg/kg/day)
111-16

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excreted slightly more nitrate than they ingested. The rats supplemented at
the higher dose (2.5 mg/kg/day) excreted slightly less nitrate than they
ingested. The possibility of a body pool of nitrate turning over during the
experiment was ruled out. After stopping supplementation no 13N03 was
detectable in the urine on the third day. The authors could not detect
nitrate in feces of conventional rats.
Schneider and Yeary (1975) studied the kinetics of the elimination of
nitrite from the plasma of seven dogs of both sexes and mixed breeds, seven
cross-bred ewes, and seven Shetland ponies of both sexes. Sodium nitrite was
administered intravenously at 20 mg/kg (4.1 mg/kg as nitrogen) and blood was
collected 15-30 seconds after injection, at 1-minute intervals between 1 and
10 minutes, at 5 minute intervals between 10 and 30 minutes, at 10 minute
intervals between 30 and 60 minutes, and then at 1.5, 2, 3, 6 and 24 hours
after injection. A colorimetric assay for nitrite was used after plasma was
isolated. The half-life for elimination of nitrite from plasma was between
0.5 and 0.6 hours in all three species. The authors concluded that this rapid
clearance indicated a process other than renal excretion, such as metabolic
conversion to nitrate.
Davison et al. (1964) added nitrate to the diets of Holstein heifers
during pregnancy. Milk samples were taken at weekly intervals from
parturition to 30 days. Milk from heifers fed normal rations averaged 5 ppm
nitrate, while milk from heifers fed 440 mg/kg/day and 660 mg/kg/day nitrate
(equivalent to 99.4 and 149.2 mg/kg/day as nitrogen) averaged 9 ppm and 15 ppm
nitrate, respectively. In contrast, Crowley et al. (1974) reported a
long-term study of a Wisconsin dairy herd drinking water with 374 ppm nitrate.
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The Holstein cows produced milk containing an average of 5.7 ppm nitrate.
This compared to controls drinking water containing 19 ppm nitrate which
produced milk containing 5.6 ppm nitrate.
E.	Bioaccumulatlon and Retention
No evidence was found for bioaccumulation of nitrate or nitrite in any
tissue storage depot. However, nitrate is retained by recycling through
saliva with reabsorption into plasma as explained in III.B. (Spiegelhalder
et al. 1976).
F.	Sugary
The primary route of nitrate and nitrite absorption is through the
gastrointestinal tract. Nitrate is absorbed by active transport through the
wall of the upper small intestine. Nitrite is absorbed by diffusion across
the gastric mucosa and also through the wall of the intestinal tract. In both
cases, absorption following ingestion is essentially complete (i.e., 100%).
Nitrate and nitrite can also be absorbed into the blood through the lungs.
Nitrate is rapidly disseminated throughout the body in the blood and is
actively secreted in saliva, gastric juices, and urine. Nitrite reacts so
rapidly with hemoglobin that little is transported to the tissues. Nitrate
and nitrite do not bioaccumulate. However, they are constantly present in the
body as a result of novo synthesis as well as ingestion.
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Nitrate is reduced to nitrite by the action of oral and gastrointestinal
bacteria. Approximately 5% of ingested nitrate is transformed to nitrite by
oral bacteria in healthy human adults and older children. Reduction of
nitrate to nitrite also occurs in the stomach of young infants and others with
abnormally high pH in the stomach. However, no quantitative data are
available on how much ingested nitrate is transformed to nitrite in the
stomach. Nitrite that is absorbed into the blood reacts with hemoglobin,
oxidizing it to methemoglobin. Nitrite in the stomach may react with
secondary amines and other amino substrates present in food or in the
epithelium to form N-nitroso compounds.
Nitrate is excreted readily through the kidneys. When excess nitrate is
not added to the diet, rats excrete significantly more nitrate than they
ingest. Nitrite is so reactive that none remains to be excreted.
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IV. HUMAN EXPOSURE
Nitrate is a normal component of the human diet. A typical daily intake
by an adult in the United States is about 75 mg/day (about 0.2-0.3 mg nitrate-
nitrogen/kg/ day) (NAS 1981). Of this, over 85% comes from the natural nitrate
content of vegetables such as beets, celery, lettuce and spinach. Daily
intakes of nitrate by vegetarians may exceed 250 mg/day (0.8 mg nitrate-
nitrogen/kg/day) (NAS 1981). The contribution from drinking water is usually
quite small (about 2%-3% of the total) (NAS 1981), but may increase to about
50% if water containing 10 mg nitrate-nitrogen is consumed (USEPA 1990). The
USEPA has performed a thorough investigation of human exposure to
nitrate/nitrite through drinking water. The findings are presented in a
document entitled, "Estimated National Occurrence and Exposure to
Nitrate/Nitrite in Public Drinking Water Supplies" (USEPA 1990).
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V. HEALTH EFFECTS IN ANIMALS
The adverse health effects of nitrate are largely due to its conversion
to nitrite. This conversion is mediated by bacteria which are present in the
alimentary tract. Thus, equal doses of nitrate often do not produce equal
effects in different species, due to differences in the number and type of
enteric bacteria. For example, ruminants are especially sensitive to nitrate
due to the high bacterial content of the rumen (Emerick 1974), while dogs
appear to reduce nitrate only slowly (Greene and Hiatt 1954). For these
reasons, studies in ruminants or dogs are not considered here, since
dose-response data are not quantitatively applicable to humans. Similarly,
dose-response data from other animal species (rats, mice, etc.) should be
extrapolated to humans with caution, due to possible differences in nitrate
reduction between species.
A. Short-Term
1. Lethality
Lethal dose data reported for nitrate and nitrite in various species are
summarized in Table V-l. As may be seen, nitrate has low acute lethality
(only slightly more than potassium or sodium chloride), and probably causes
death mainly by electrolyte imbalances (ECETOC 1988). In contrast, nitrite
causes lethality at about 1/10 the dose of nitrate. This is presumably due to
either massive methemoglobinemia and/or severe hypotension (see below). It is
V-l

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Table V-l Oral LDJ0 of Nitrate arid Nitrite
Animal
Cation
LDjo for
no3-
LD50 for
NOz"
Re fere
mg N03/kg
mg N/kg
mg NOz/kg
mg N/kg
Rat
Na+


100
30
a
Rat
Na+


120
36
b
Rat
K+
1,986
457


c
Rat
K+


46
14
c
Rat
Na+


105
30
d
Rat (1-yr)
Na+


51-87
15-26
e
Rat (3-mo)
Na+


73
22
e
Mouse
K+


119
36
c
Mouse
K+


95
28
c
Rabbit
K+
1,166
268
108
32
b
Rabbit
Na+
1,955
450


b
aIntaizumi et al. 1980
'Merck Index 1976
TfflO 1962
dImaizumi et al. 1980
"Druckrey et al. 1963
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important to note that these LD50 values refer to large single doses of
compound, and that administration of equal or even greater doses spread out
over the course of a day in food or water do not result in lethality (Shuval
and Gruener 1977, Imaizumi et al. 1980).
2. Methemoglobinemia
Nitrate
No short-term studies were located on the effect of nitrate ingestion on
methemoglobinemia levels in animals.
Nitrite
Shuval and Gruener (1977) administered single oral doses of 0, 2.5, 5,
10, 15, 20, 25 or 30 mg/kg of NaN02 to pregnant rats. This corresponds to
doses of 0, 0.5, 1, 2, 3, 4, 5 or 6 mg nitrite-nitrogen/kg/day. There was a
clear dose-response increase in methemoglobin levels in the dam, ranging from
0.9% in controls to 60% in animals given 30 mg/kg NaN02. Methemoglobin levels
in the fetuses also increased, but were about 50% those of the dam.
Shuval and Gruener (1977) supplied groups of fifteen 50-day-old male
C57bl/6J mice with drinking water containing 0, 100, 1,000, 1,500 or 2,000 mg
sodium nitrite/L for 3 weeks. These levels produced doses of 0, 2.6, 26, 40
or 53 mg nitrite-nitrogen/kg/day. No change in methemoglobin level was
observed at 2.6 mg nitrite-nitrogen/kg/day. An increase was observed in
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animals receiving 26 mg nitrite-nitrogen/kg/day, but this was not
statistically significant. Doses of 40 and 53 mg nitrite-nitrogen/kg/day both
produced a significant increase in methemoglobin levels (P<0.05).
Shuval and Gruener (1972, 1977) supplied groups of 12 pregnant rats
(strain "sabra") with water containing 0, 2,000 or 3,000 mg/L sodium nitrite
through the 3rd week after parturition. This corresponded to doses of 1, 54
or 81 rag nitrite-nitrogen/kg/day. Mean methemoglobin values were 1.1%, 5.5%
and 24% for the females in the control, low-dose, and high-dose groups,
respectively. From birth to weaning, pups in the treated groups showed no
abnormally high methemoglobin, but total hemoglobin was 20% lower than
controls.
Imaizumi et al. (1980) studied the effect of sodium nitrite on
methemoglobin formation in rats. Sprague-Dawley rats (200 g) were given
single oral doses of NaN02 by gastric intubation at dose levels of 0, 10, 25,
50, 100 or 150 mg/kg (equivalent to 0, 2, 5, 10, 20 or 30 mg nitrite-
nitrogen/kg). Within 1 hour after administration of the high dose,
methemoglobin concentrations increased to 45%-80%. Methemoglobin levels
subsequently returned to control levels (approximately 2%) at 24 hours. A
clear dose-response relationship was apparent between nitrite dose and
methemoglobin levels, with maximum levels of 10%, 40%, 70%, and 80% at doses
of 25, 50, 100 and 150 mg/kg, respectively.
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3. Effects of Blood Pressure
Nitrite ion is a smooth muscle relaxant that can cause vasodilation and
hypotension. No studies were located on vasodilation following oral exposure
to nitrite ion, but several studies investigated hypotensive effects following
intravenous injection of NaN02. For example, Constantine et al. (1971)
injected 12 dogs with 4 mg/kg of NaN02 (0.8 mg nitrite-nitrogen/kg), and
observed an average decrease in blood pressure of 26 mnHg. Similarly, Klimmek
et al. (1983) injected 5 dogs with 15 mg/kg of NaNOz (3 mg nitrite-
nitrogen/kg/ day) , and observed an average increase of 50% in blood flow to the
brain (indicative of cerebral vasodilation). Similar effects are produced by
organic nitrates (e.g., aunyl nitrate, nitroglycerin), but not by inorganic
nitrate ion (Nickerson 1975).
4. Histopathological Effects
Nitrate
No studies were located regarding histopathological effects resulting
from short-term ingestion of nitrate by animals.
Nitrite
Inai et al. (1979) exposed groups of 10 male and 10 female mice to NaN02
in drinking water for 6 weeks. Concentration levels were 0, 500, 1,250, 5,000
or 10,000 mg N03/L (corresponding to doses 0, 15, 37, 150 or 300 mg nitrite -
nitrogen/kg/day). Microscopic examination of visceral organs revealed slight
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degeneration and spotty necrosis of hepatocytes In liver, along with
hemosiderin deposition in liver, spleen and lymph nodes. This was judged to
be secondary to a hemolytic effect of NaN02. No data were provided regarding
dose-response relationships for these effects.
Nitrite plus Nitrosatable Substrates
Asahina et al. (1971) studied the acute toxicity of sodium nitrite and
dimethylamine in adult male Charles River mice. Liver necrosis was observed
when 1,000 mg/kg dimethylamine and 100 mg/kg sodium nitrite (20.3 mg nitrite -
nitrogen/kg) were given simultaneously by gavage. The necrosis was attributed
to intragastric formation of nitrosodimethylamine. Liver injury was reduced
when the nitrite was given 15-120 minutes before, but not 3 hours after, the
dimethylamine. This suggests that nitrite disappeared fairly rapidly from the
stomach.
Lijinsky and Greenblatt (1972) exposed rats by gavage to 1.0 mL doses of
aiainopyrine (35 mg/mL), NaN02 (5-40 mg/mL) or both aminopyrine (35 mg/mL) and
nitrite (5-40 mg/mL). These doses of NaN02 correspond to 3-24 mg nitrite-
nitrogen/kg/day. After 48 hours, animals were killed and the livers examined
histologically. There was a clear dose-dependent increase in the occurrence
of hepatic necrosis as a function of nitrite level in the presence of
aminopyrine, while no effect was observed following exposure to aminopyrine
(35 mg/mL) or NaN02 (40 mg/mL) alone. The reaction of nitrite plus
aminopyrene is known to yield dimethylnitrosaaine io vitro (Lijinsky 1974),
and Lijinsky and Greenblatt concluded that the hepatic effects were the result
of dimethylnitrosanine formation is vivo.
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Acute nitrosamine toxicity in the liver has been prevented by
simultaneous administration of ascorbic acid with the nitrite and amine
(Greenblatt 1973, Kamm et al. 1975). Ascorbate prevents toxicity only when it
is present in the stomach of the rat with the amine and nitrite. Erythorbic
acid (Kamm et al. 1975) and an emulsion of a*tocopherol (vitamin E) (Kamm
et al. 1977) are as effective as ascorbic acid in preventing hepatic necrosis.
Complete protection from measurable liver toxicity (elevation of SGPT)
required that the inhibitor be at least equimolar with the nitrite (Kamm
et al. 1977). Prevention of histologically observed necrosis required twice
the amount of ascorbic acid (Greenblatt 1973). The vitamins are oxidized in
the process of inactivating nitrite. The common antioxidants BHA (butylated
hydroxyanisole) and BHT (butylated hydroxytoluene) had no measurable
protective value at 0.6 equimolar (Astill and Mulligan 1977).
5. Effects on Thyroid Function
Wyngaarden et al. (1953) studied the effects of high levels of nitrate in
drinking water on the iodide-concentrating mechanism of the thyroid gland.
Nitrate appeared to compete with iodide in uptake by the thyroid, leading to a
decreased iodide uptake. Horing et al. (1985) and Seffner et al. (1985) (as
reported in ECETOC 1988) exposed rats to drinking water containing 40, 200,
1,200 or 4,000 mg/L of nitrate for 100 days. This corresponds to doses of
0.9, 4.6, 28 or 92 mg nitrate-nitrogen/kg/day. Thyroid function was examined
by 131I-uptake, mI-serum level, 131I-corporation into proteins, the thyroid
gland weight and histopathological parameters. Thyroid weight and 131I-uptake
V-7

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appeared to be changed slightly at all dosage levels. Histopathological
parameters were altered at all dose levels but there was no dose-response
relationship in the finding.
Jahreis et al. (1987) investigated the effect of nitrate on thyroid
hormone production in young pigs. Groups of 7 animals were exposed to 0 or 3%
KN03 in the diet for 2 days. Assuming consumption of 0.04 kg food/kg body
weight per day, this corresponds to a dose of 170 mg nitrate-nitrogen/kg/day.
Animals exposed to KN03 had significantly reduced serum levels of (P<0.01),
but not of Tj. Supplementation of the diet with excess iodide (0.5 mg/kg)
resulted in increased levels of Tt in both groups, and the difference between
them was not significant. Similar results were described when exposure was
continued for 6 weeks, although excess iodide was not able to fully reverse
the effect of nitrate. The authors interpreted these observations to show
that nitrate inhibited synthesis of thyroid hormones. However, since T3 is
the biologically active form of thyroid hormone, and since T3 levels were not
affected by nitrate exposure, the relevance of these observations is not
clear.
B. Longer-Term
1. Methemoglobinemia/Hematology
Nitrate
Shuval and Gruener (1977) supplied groups of 52 male rats with water
containing 0 or 2,000 mg/L of NaNOs (0 or 330 mg/L nitrate-nitrogen) for
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16 months. This corresponded to average daily doses of 150-300 mg N03/kg/day
(34-69 mg nitrate-nitrogen/kg/day). Mean methemoglobin levels were not
significantly elevated at any time between 1 and 16 months. This study
identifies a NOAEL of 69 mg nitrate-nitrogen/kg/day.
Chow et al. (1980) provided young male rats (8/group) with drinking water
containing 0 or 400 mg sodium nitrate/L (equivalent to 0 or 6.6 mg nitrate-
nitrogen/kg/day) . Rats received this treatment for 16 weeks during which time
blood samples were collected for the evaluation of methemoglobin.
Methemoglobin concentrations fluctuated between 0 and 1.2%. Although no
statistical evaluation was reported, this was the sane range of concentrations
reported for controls. This study identifies a NOAEL of 6.6 mg nitrate-
nitrogen/kg/day.
Chow et al. (1980) studied the effects of chronic nitrate exposure on
methemoglobin concentration in male Sprague-Dawley rats. Rats were given 0 or
4,000 mg sodium nitrate/L (an amount equivalent to 0 or 66 mg nitrate-
nitrogen/kg/day) for 14 months. Methemoglobin concentration, measured every
other week throughout the course of the study, fluctuated between 0 and 2%.
This was not significantly elevated over control values. This study
identifies a NOAEL of 66 mg nitrate-nitrogen/kg/day.
Nitrite
Druckrey et al. (1963) exposed 30 male and 30 female rats to NaN02 in
drinking water at a level that resulted in ingestion of 100 mg N02/kg/day
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(20 mg nitrite-nitrogen/kg/day). Exposure was for 70 days. Methemoglobin
levels averaged 7% In the exposed animals compared to 0.8% in controls.
Shuval and Gruener (1972) exposed groups of 8 male rats for 24 months to
water containing 0, 100, 1,000, 2,000 or 3,000 ppm of sodium nitrite. This
corresponds to doses of about 0, 2, 20, 40 or 60 mg nitrite-nitrogen/kg/day.
There were no significant differences in growth or mortality between the
groups. Methemoglobin levels were increased to 5%, 12% and 22% in the 1,000,
2,000 and 3,000 ppm dose groups, respectively.
Chow et al. (1980) provided young male rats (8/group) with drinking water
containing 0 or 200 mg sodium nitrite/L (equivalent to 0 or 4 mg nitrite -
nitrogen/kg/day). Rats received this treatment for 16 weeks during which time
blood samples were collected for the evaluation of methemoglobin.
Methemoglobin concentration fluctuated between 0.5% and 3.1% compared to
control methemoglobin values of less than 1.2%. Statistical evaluation of
these data was not reported.
Chow et al. (1980) measured the effects of chronic nitrite exposure on
methemoglobin concentration in male Sprague-Dawley rats. Rats were provided
with drinking water containing 0 or 2,000 mg sodium nitrite/L (equivalent to 0
or 40 mg nitrite-nitrogen/kg/day) for 14 months. Blood was analyzed for
methemoglobin every other week throughout the course of the study.
Methemoglobin in nitrite-treated rats fluctuated from 1% to 35%, and was
significantly greater than the methemoglobin concentration (1%) in animals
receiving water. This study identifies a L0AEL of 40 mg nitrite-
nitrogen/kg/day but does not identify a NOAEL.
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Imaizumi et al. (1980) studied the effects of long-term ingestion of
sodium nitrite on methemoglobin concentration and other hematological
parameters in the rat. Four Sprague-Dawley rats (200 g) were given drinking
water containing 0 or 5,000 mg/L NaN02 for 6 months. The average daily intake
of NaN02 was 50 mg nitrite-nitrogen/kg/day. Fluctuations in methemoglobin
occurred as a function at the time of sampling, a phenomena attributed to the
nocturnal water consumption patterns of the rat. Maximum methemoglobin
concentrations (ranging from 33% to 88%) were observed between 6:00 and
9:00 p.m., and minimum concentrations (ranging from 4% to 19%) at 9:00 a.m.
The 6 month average methemoglobin level measured at 3:00 p.m. was 8.2%
compared to 1.0% in control rats. No abnormalities in growth or development
were observed in the treated group compared to controls. Although
hematological effects other than methemoglobinemia (erythrocytic Heinz bodies,
anisocytosis and hypohemoglobinemia) were seen in the treated rats, the extent
of these effects were not described.
Til et al. (1988) investigated the subchronic oral toxicity of potassium
nitrite in rats. Groups of 10 males and 10 females were given drinking water
containing 0, 100, 300, 1,000 or 3,000 mg/L of KN02 for 13 weeks. This
corresponds to doses of 0, 1.7, 5, 17 or 50 mg nitrite-nitrogen/kg/day. All
solutions were adjusted (by addition of KC1) so that each contained the same
final concentration of potassium (35 mM). Methemoglobin levels increased from
1.7%-2.3% in the control group to 5.7%-7.6% in the high-dose group (P<0.05).
No significant increases in methemoglobin were noted at lower doses. This
study identifies a NOAEL of 17 and a LOAEL (based on methemoglobin) of
50 mg nitrite-nitrogen/kg/day.
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Grant and Butler (1989) administered NaN02 to groups of 50 male F344 rats
for 115 weeks. Dose levels were 0, 0.2 or 0.5% in the diet, corresponding to
intakes of about 20 or 50 mg nitrite-nitrogen/kg/day. Transient effects were
observed at both dose levels for a number of hematological parameters,
including decreased red blood cell counts, mean red cell volume, hematocrit
and hemoglobin concentration. These effects developed slowly over the first
8 weeks of the study, then tended to return to near normal by 52 weeks.
Methemoglobin levels were not measured. Based on the changes in blood, this
study identifies a chronic oral LOAEL of 20 mg nitrite-nitrogen/kg/day. A
NOAEL was not identified.
2. Histopathological Effects
Nitrate
Sugiyama et al. (1979) fed groups of 50 male and 50 female mice sodium
nitrate at dose levels of 0, 2,500 or 5,000 mg/kg/day (0, 410 or 820 mg
nitrite-nitrogen/kg/day) for more than a year. Amyloidosis was present in 37%
of survivors at the high dose, in 42% at the lower dose, and in 25% of the
controls. The authors reported that the liver was more affected by sodium
nitrate than other organs and showed marked atrophy and hemosiderosis.
Amyloidosis, however, was noted in various organs including the liver, spleen,
kidneys and adrenals. Quantitative data on these lesions were not reported.
Chow et al. (1980) provided young male rats (8/group) with drinking water
containing 0 or 400 mg sodium nitrate/L (equivalent to 0 or 6.6 mg nitrate-
nitrogen/kg/ day) . No animal mortality occurred during the course of
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treatment. At the completion of 16 weeks of treatment, rats were sacrificed
and histologically examined. Liver, kidney, spleen, heart and body weights
were not altered in treated animals. Lung weights in the nitrate-treated rats
averaged 1.7 g compared to 1.5 g in control rats. No other statistical or
histopathologic details were reported.
Chow et al. (1980) measured the effects of chronic nitrate exposure on
histopathology in male Sprague-Dawley rats. Rats were provided drinking water
containing 0 or 4,000 mg sodium nitrate/L (equivalent to 0 or 66 mg nitrate-
nitrogen/kg/day) for 14 months. Four of the 10 rats given nitrate for
14 months survived. Nitrate-treated rats had lighter livers and heavier lungs
than the control group, and moderate lung lesions were observed in all of the
surviving rats. This study identifies a L0AEL of 66 mg nitrate-
ni trogen/kg/day.
Fritsch et al. (1980) supplied groups of ten male Sprague-Dawley rats
with a diet containing 0, 0.5 or 5% nitrate for 6 months. This corresponds to
doses of about 0, 50 or 560 mg nitrate-nitrogen/kg/day. Changes were observed
in the spleen in all the treated animals. The changes included a significant
release of ferric iron and the presence of hemorrhagic areas. Further details
on the magnitude or severity of these changes were not provided. These
changes may be secondary to the hematological effects of nitrite.
NUrUg
Druckrey et al. (1963) exposed 30 male and female rats for their lifetime
to NaN02 in drinking water at a dose rate of 100 mg/kg/day (20 mg nitrite-
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nitrogen/kg/day). The lifespan of the created animals was decreased to an
average of 630 days as compared to 730 days for the control animals. Body
weights for the treated rats were 11% below those of the controls. This was
attributed to a decreased willingness of the exposed rats to drink the water.
No treatment-related histopathological effects were noted in liver, kidney,
lungs or spleen.
Shuval and Gruener (1972) exposed groups of 8 male rats for 24 months to
water containing 0, 100, 1,000, 2,000 or 3,000 ppm of sodium nitrite. This
corresponds to doses of about 0, 2, 20, 40 or 60 mg nitrite-nitrogen/kg/day.
There were no significant differences in growth or mortality between the
groups. Histological examination of the lungs revealed dilated bronchi and
the mucosa and muscles were often atrophied. Interstitial fibrosis and
emphysema were also observed. These changes were found with increasing
frequency and severity in the 1,000, 2,000 and 3,000 ppm groups. Histological
examination of the heart revealed an increased percentage of coronary arteries
that were characterized as "thin and dilated." Similar effects on the
coronary arteries were reported in a follow-up study by Shuval and Gruener
(1977) at exposure levels of 200, 1,000, 2,000 or 3,000 mg/L of NaN02.
Exposure to 2,000 ng/L of NaN0} also produced the saae effect. However, it is
not clear how many samples or how many vessels were examined from each animal,
so the LOAEL for this effect cannot be defined from the data presented. Also,
this effect appears to be at least partly due to the absence of coronary
artery thickening and narrowing that normally occurs in aged rats, so it is
not certain that these changes are inherently adverse. These studies identify
a N0AEL of 2 and a LOAEL (based on effects on the lung) of 20 mg nitrite-
nitrogen/kg/day .
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Inai et al. (1979) exposed groups of 50 male and 50 female mice to
drinking water containing 0, 1,250, 2,500 or 5,000 mg sodium nitrite/L (0,
41.6, 83.2 or 166.6 mg nitrite-nitrogen/kg/day) for more than 18 months.
Histological examinations revealed marked atrophy and hemosiderosis in liver,
and amyloidosis in liver, spleen, kidneys and adrenal. However, no data were
presented on the doses which caused these effects.
Chow et al. (1980) provided young male rats (8/group) with drinking water
containing 0 or 200 mg sodium nitrite/L (equivalent to 0 or 4 mg nitrite-
nitrogen/kg/day). At the completion of 16 weeks of treatment, rats were
sacrificed and histologically examined. No animals deaths occurred as a
result of the treatment. The lungs of rats given nitrite averaged 2.0 g
compared to the average lung weight of 1.5 g of control rats. The authors did
not report whether this difference was statistically significant. Liver,
heart, kidney, spleen and body weight were not affected by treatment with
nitrite. No other histopathological details were reported.
Chow et al. (1980) measured the effects of chronic nitrite exposure on
histopathology in male Sprague-Dawley rats. Rats were provided drinking water
containing 0 or 2,000 mg sodium nitrite/L (equivalent to 0 or 40 mg nitrite-
nitrogen/kg/day) for 14 months. Surviving animals (5/12) were found to have
significantly increased lung weights when compared to controls or animals
given nitrate. Examination of lung tissue revealed chronic pneumonitis
(microabscesses, congestion) in all nitrite-exposed rats. This study
identifies a LOAEL of 40 mg nitrite-nitrogen/kg/day but does not identify a
NOAEL.
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Fritsch et al. (1980) supplied groups of ten male Sprague-Dawley rats
with a diet containing 0, 0.05 or 0.5% Nafl02 for 6 months. This corresponded
to doses of 0, 6.3 or 74 mg nitrite-nitrogen/kg/day. Changes were observed in
the spleen in all the treated animals. The changes included a significant
release of ferric iron and the presence of hemorrhagic areas. Further details
were not provided on the magnitude or the severity of these changes.
Til et al. (1988) investigated the subchronic oral toxicity of potassium
nitrite in rats. Groups of 10 males and 10 females were given drinking water
containing 0, 100, 300, 1,000 or 3,000 mg/L of KN0Z for 13 weeks. This
corresponds to doses of 0, 1.7, 5.0, 17 or 50 mg nitrite-nitrogen/kg/day. All
solutions were adjusted (by addition of KC1) so that each contained the same
final concentration of potassium (35 mM). Water intake tended to decrease in
a dose-dependent fashion, although this was statistically significant only in
the two highest doses. Complete histological examination revealed no
pathological changes in any tissue at any dose, except for a very-slight to
slight hypertrophy of the zona glomeruloa in animals supplied 300 mg/L or
higher. It seems likely that this response is a normal endocrine adaptation
to a change in blood volume and/or blood pressure resulting from the decreased
water intake, and is not judged to constitute an adverse health effect,
3. Other Effects
Ten male Sprague-Dawley rats initially weighing 50 grams were fed
2,500 mg/kg/day sodium nitrate (412.5 mg/kg/day nitrate-nitrogen) in the diet
for 6 months. The results showed a statistically significant (P<0.01)
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diuretic effect by 2 months when compared to rats fed equimolar sodium
chloride. Rats fed one-tenth of that dose showed no significant diuresis
(Fritsch et al. 1980).
C. Reproductive/Teratogenicity Effects
Nitrate
The U.S. Food and Drug Administration sponsored extensive tests of the
reproductive and teratogenic effects of NaNOj and KN03 in mice, rats, hamsters
and rabbits (FDA 1972a, b). Groups of 20-26 mice, rats or hamsters and 10-13
rabbits were treated by gavage on days 6-15 (mice, rats), days 6-10 (hamster)
or days 6-18 (rabbits) of gestation. Fetuses were delivered by Cesarean
section, and examined for visceral and skeletal malformations. Dose levels
(expressed as nitrate-nitrogen) ranged from 0.6 to 66 mg/kg/day for mice, from
0.3 to 41 mg/kg/day for rats, from 0.4 to 66 mg/kg/day for hamsters and from
0.3 to 41 mg/kg/day for rabbits. No significant effects were detected
regarding maternal reproductive parameters (percent pregnant, abortion
frequency, number of litters), fetotoxicity (percent fetal resorptions, live
fetuses per dam, average fetal weight) or fetal malformations up to the
maximum doses administered to each species. These studies identify a
reproductive/developmental NOAEL of 66 mg nitrate-nitrogen/kg/day for mice and
hamsters and 41 mg nitrate-nitrogen/kg/day for rats and rabbits.
Sleight and Atallah (1968) studied the effects of nitrate on reproduction
and development in guinea pigs. Groups of 3-6 females were exposed to
drinking water containing 0, 300, 2,500, 10,000 or 30,000 ppm KN03 for
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143-204 days. Based on measured water intake and body weight, this resulted
in average doses of 0, 12, 102, 507 or 1,130 mg nitrate-nitrogen/kg/day.
Normal conception occurred at all dose levels. Since males were also exposed,
their fertility was not impaired. No statistically significant effect on
reproductive performance (litters produced, number of live births) was
detected except in the high dose group, where there was a decrease in number
of live births (only 2 from 3 females, compared with 31 from 4 females in the
control group). Statistical significance was not reported. One female in
this group died, and had 4 mummified fetuses in utero. The authors attributed
the fetal effects to hypoxia due to maternal methemoglobinemia, although data
on this were not provided. No fetotoxic malformations were observed at any
dose. This study identifies a reproductive NOAEL of 507 and a LOAEL of
1,130 mg nitrate-nitrogen/kg/day.
Alavantic et al. (1988b) exposed male C3H X 101 Fi mice (5/group) to 0,
600 or 1,200 mg/kg/day sodium nitrate (99 or 198 mg nitrate-nitrogen/kg/day)
by stomach intubation for 3 days. Mice were sacrificed 11 and 17 days
following treatment and 500 sperm cells per animals from the caudal epididymis
were examined for abnormal morphology. No increase in sperm-head
abnormalities were observed in mice receiving sodium nitrate at 600 mg/kg/day.
A slight increase (not significant) was noted in mice receiving
1,200 mg/kg/day. This study identified a NOAEL for sodium nitrate of
600 mg/kg/day for sperm-head abnormally in mice.
Alavantic et al. (1988a) investigated the effect of sodium nitrate on
reproduction in mice. Hale and female C3H X 101 mice (10-12 weeks old, 25 per
group) were dosed intragastrically with 600 or 1,200 mg/kg/day NaN03 (99 or
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198 mg nitrate-nitrogen/kg/day) daily for 14 days prior to mating. Fertility,
litter size and sex ratio were evaluated and the degree of heritable
translocations and sperm abnormalities were examined in both parents and
offspring. Fertility and litter size were not reduced in the mice receiving
either 600 or 1,200 mg/kg/day NaN03. Cytogenetic evaluation of the
spermatocytes of parental mice revealed a significant increase in sex
chromosome univalency at both doses. Spern-head abnormalities were
significantly increased in mice receiving 1,200 mg/kg/day but not at the low
dose group. No effect was evident at either dose level on testes weight or
body weight. Examination of Fj^ males revealed no heritable chromosomal
translocations, induction of sex chromosomal univalency, or increase in the
number of sperm-head abnormalities at either dose. This study identifies a
LOAEL of 600 mg/kg/day NaN03 for chromosomal changes in mouse germ cells.
Markel et al. (1989) investigated the effect of nitrate exposure on the
development of sensory-motor function and learning behavior in the rat.
Drinking water containing 0, 1.12 or 2.24 mM KN03 (120 or 240 mg KN03/L) was
provided to pregnant Wistar rats through gestation and lactation. This
corresponds to doses 0, 1.7 or 3.4 mg nitrate-nitrogen/kg/day. At 21 days
postpartum (weaning), the offspring were also provided the nitrate-containing
water. Sensory-motor parameters evaluated included righting reflex and
cliff-avoidance, open and closed-field locomotor behavior, homing behavior,
eye-opening and startle. Behavioral parameters included one-way active
avoidance, and water rewarded discrimination learning. Nitrate-exposed rats
developed mature righting and cliff-avoidance reflexes and hearing startle
reaction earlier than controls. Motor activity (open field) was increased
soon after birth in treated animals but at day 20 hypoactivity was reported.
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As adults, created rats showed deficits in rewarded discriminitive learning
tests and in establishing active avoidance response (P<0.001). This study
identifies a LOAEL of 1.12 mM KN03 (17 mg nitrate-nitrogen/L) for
neurobehavioral development in the rat, although the biological significance
of the observed changes are not certain.
Nitrite
The U.S. Food and Drug Administration sponsored extensive tests of the
reproductive and teratogenic effects of NaN02 and KN02 in mice, rats, hamsters
and rabbits (FDA 1972c,d). Groups of 20-26 mice, rats or hamsters and 10-13
rabbits were treated by gavage on days 6-15 (mice, rats), days 6-10 (hamster)
or days 6-18 (rabbits) of gestation. Fetuses were delivered by Cesarean
section, and examined for visceral and skeletal malformations. Dose levels
(expressed as mg nitrite-nitrogen) ranged from 0.04 to 4.6 mg/kg/day for mice,
from 0.1 to 2 mg/kg/day for rats, from 0.04 to 5.3 mg/kg/day for hamsters and
from 0.03 to 4.6 mg/kg/day for rabbits. No significant effects were detected
regarding maternal reproductive parameters (percent pregnant, abortion
frequency, number of litters), fetotoxicity (percent fetal resorptions, live
fetuses per dam, average fetal weight) or fetal malformations up to the
maximum doses administered to each species. In rats, the highest dose tested
produced slight indications of delayed skeletal maturation, especially with
respect to ribs and skull. In hamsters, a delay in skeletal maturation, which
was not clearly dose related, was observed. These effects are not considered
to be of practical health significance. These studies identify a
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reproductive/developmental NOAEL of 5.3 mg nicrice-nicrogen/kg/day for mice
and hamsters, 4.6 mg nitrite-nitrogen/kg/day for rabbits, and 2.0 mg nitrite-
nitrogen/kg/day for rats.
Druckrey et al. (1963) supplied rats with NaN02 in drinking water for
three generations at a dose level of 100 mg/kg/day (20 mg nitrite-
nitrogen/kg/day) . No teratogenic effects or adverse effects on reproduction
were detected in any generation.
Sleight and Atallah (1968) studied the effects of nitrite on reproduction
and development in guinea pigs. Groups of 3-6 females were given water
containing 0, 300, 1,100, 2,000, 3,000, 4,000, 5,000 or 10,000 ppm of KN02 for
100-240 days. Based on measured water intakes and body weights, this
corresponded to average doses of 0, 18, 45, 154, 182, 192, 244 or 577 mg
nitrite-nitrogen/kg/day. Reproductive performance was relatively normal at
exposure levels up to 3,000 ppm, but was severely affected at exposure levels
of 4,000 ppm or higher. No live births occurred in animals given 5,000 or
10,000 ppm. This study identifies a reproductive NOAEL of 182 and a L0AEL of
192 mg nitrite-nitrogen/kg/day.
Shuval and Gruener (1972, 1977) studied the effects of sodium nitrite on
growth and postnatal survival in rats. Groups of 12 pregnant rats (strain
"sabra") were given water containing 0, 2,000 or 3,000 mg/L sodium nitrite
through the third week after parturition. This corresponded to doses of 0, 54
or 81 mg nitrite-nitrogen/kg/day. Birth weights were similar in all groups.
After birth, pups of treated dams lagged in growth rate. At the end of
21 days, mean weight of the control group was 51.5 g compared to 29.5 g and
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18.5 g in the low- and high-dose treated groups, respectively. Neonatal
mortality during the lactational period was 6%, 30% and 53% in the control,
low dose and high dose groups, respectively.
Globus and Samuel (1978) studied the effect of nitrite exposure during
pregnancy on the fetal hematopoietic tissues in CD-I mice. Groups of
36-39 mice were intubated with 0 or 16.7 mg/kg/day of NaN02 (3.4 mg nitrite-
nitrogen/kg/day) from day 0 until sacrifice. The treated and control mice
were divided into subgroups that were sacrificed on the 14th, 16th or 18th day
of gestation. Analysis of fetal livers indicated that maternally administered
sodium nitrite stimulated fetal hepatic erythropoiesis. After removal of
viscera, the fetuses were fixed and stained for skeletal examination. Fetal
mortality, fetal resorption, the mean number of offspring per litter, the mean
weight per fetus, and the incidence of skeletal malformations were not
significantly different from controls (F value not stated).
Hugot et al. (1980) performed a three-generation study in rats. Groups
of 66 female animals were administered 0, 1,500 ppm or 3,000 ppm NaN02 in the
diet. These levels are equivalent to 90 or 160 mg nitrite-nitrogen/kg/day.
There were no effects on the percentage of matings resulting in pregnancy, the
average of implanted embryos, the frequency of resorptions, the percentage of
pregnancies resulting in the birth of live litters, the sex ratio or the
percentage of born animals that survived 3 days. Birth weight was slightly
lower in Flb and F2b pups in the group fed 3,000 ppm. After birth, the rate of
weight gain by pups of the 1,500 ppm group and the 3,000 ppm group lagged
behind the control group by 10% and 30%, respectively. This effect was most
prominent in the F2. and F2b groups. In the treated groups, changes in various
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organ weights were observed In a high proportion of pups at weaning. These
included increased liver, kidney and heart weights and decreased spleen and
thymus weights. Histological studies were done on F2b pups only. The main
abnormalities observed in these weanlings were fatty degeneration of the liver
and congestion of the spleen. The authors concluded that the effects of
nitrite ingestion on fertility and gestation in the rat were very limited, but
that the body and organ growth of the pups were affected between birth and
weaning. This study identifies a LOAEL of 90 mg nitrite-nitrogen/kg/day.
Olsen et al. (1984) fed a diet containing potassium nitrite-treated meat
to 70 male and 140 female Wistar rats. The nitrite content of the diet was
reported to be 2, 4 or 94 mg/kg (corresponding to doses of 0.02, 0.04 or
0.9 mg nitrite-nitrogen/kg). Rats were fed this diet for 10 weeks prior to
mating. No differences in pregnancy rate, litter size, mean pup weight or
survival were observed between treated and control rats. No teratogenic
%
effects were reported.
Vorhees et al. (1984) investigated the developmental toxicity of sodium
nitrite in rats. Groups of males (numbers not specified) and females
(22-32/dose group) were given food containing 0, 0.0125, 0.025 or 0.05% NaN02
for 14 days prior to breeding and during breeding (14 days). Exposure of
females was continued during gestation (22 days) and lactation (21 days).
After weaning, offspring were supplied HaN02 in the diet at the same level as
their parents for 90-180 days. No effects were observed for a number of
reproductive parameters, including number of females with sperm, percentage of
females with spera delivering, number of litters with more than 8 pups,
gestational lengths, number of pups per litter and male/female ratio. No
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malfornations were detected. However, NaN02 produced a statistically
significant increase in preweaning mortality and a decrease in pup body weight
in the middle and high dose groups. Pups were subjected to a number of
neurobehavioral tests. Most tests were negative, but delays in swimming
development were detected in the middle and high dose group during lactation.
Decreased open field activity was also noted, but this was not dose dependent.
The authors interpreted the results to reflect a delay in development rather
than specific central nervous system damage. This study identifies a NOAEL of
0.0125% and LOAEL of 0.025% in food. This corresponds to doses of about 1.2
and 2.5 mg nitrite-nitrogen/kg/day to the dam.
Roth et al. (1987) supplied groups of 8-10 females Long-Evans hooded rats
with water containing 0, 2,000 or 3,000 mg/L of NaN02 throughout gestation and
lactation. Based on measured water intake rates, the average daily exposures
were 44 or 60 mg nitrite-nitrogen/kg/day during gestation and 84 or 103 mg
nitrite-nitrogen/kg/day during lactation. No evidence of maternal toxicity
was described. No significant differences in litter size, pup weight or sex
ratio were observed at birth between pups bom to treated vs untreated dams.
However, pups born to treated dams did not gain weight as well as controls and
pup mortality at day 21 was increased in both dose groups. The affected pups
were pale, weak and had distended abdomens. At day 9, pups from the low and
high-dose groups were anemic, with hemoglobin levels of 6.70 g/dL and
6.13 g/dL respectively, compared to 9.85 g/dL in controls. The anemia became
somewhat more severe by day 16. Blood smears revealed hypochromasia and
anisocytosis in treated pups. Methemoglobin was not elevated in either
treatment group compared to controls.
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Roth et al. (1987) supplied groups of 5-8 female Long-Evans hooded rats
(180-200 g) with drinking water containing 0, 500, 1,000 or 2,000 mg/L of
NaN02 throughout gestation and lactation. This corresponds to doses of 0, 10,
20 or 40 mg nitrite-nitrogen/kg/day. Maternal weight gain during gestation
did not differ between groups. Selected pups were sacrificed and blood
samples collected 7, 9, 13, 16 and 20 days postpartum. Dose dependent
decreases were noted in hemoglobin concentration, red blood cell count and
mean corpuscular volume. Of these, mean corpuscular volume appeared to be the
most sensitive indicator of effect. Statistically significant decreases were
noted in the two highest dose groups on all days treated. This study
identifies a NOAEL of 10 and a LOAEL of 20 mg nitrite-nitrogen/kg/day.
Roth et al. (1987) provided pregnant Long-Evans hood rats with water
containing 0 or 2,000 mg NaNOz/L during gestation. This corresponds to doses
of 0 or 40 mg nitrite-nitrogen/kg/day. Following birth, the pups were
separated from their maternal dams and were distributed to foster dams
(10 pups/dam) as follows: control pups (mothers given tap water) to control
dams (5 litters); exposed pups to control mothers (6 litters); control pups to
exposed dams (5 litters); and exposed pups to exposed dams (5 litters).
Selected pups were killed on days 7, 14 and 21 for hematological and
histological examination. No significant differences were observed between
exposed and control groups with respect to pup weight, litter size, sex ratio
or postpartum mortality. Pups suckled by dams exposed to sodium nitrite in
their drinking water weighed significantly less than pups suckled on dams
drinking tap water. This effect was seen regardless of prenatal treatment
received by the pups. Anemia was also observed in pups by day 14 suckled on
dams exposed to nitrite-containing water. Red blood cell counts, mean
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corpuscular volume and hemoglobin were significantly reduced in these pups.
These effects were not observed in pups exposed only during gestation. The
authors concluded that post-natal exposure (via the milk during lactation) was
more important in the anemic response than was exposure during gestation.
This study identifies a LOAEL of 40 mg nitrite-nitrogen/kg/day, but does not
identify a NOAEL.
Shiobara (1987) administered doses of 0, 20, 40, 80 or 120 mg/kg/day of
NaN02 to pregnant mice (25/dose) on days 6-15 of gestation. This corresponds
to doses of 0, 4, 8, 16 or 24 mg nitrite-nitrogen/kg/day. Fetuses were
examined on day 17 for external and skeletal malformations. In the two
highest dose groups, females gained less weight than normal, and the number of
implants, living fetuses and litter size were all significantly decreased. No
treatment-related teratogenic effects were detected. This study identifies a
NOAEL of 8 and a LOAEL of 16 mg nitrite-nitrogen/kg/day.
Alavantic et al. (1988b) exposed groups of 5 male C3H X 101 mice to 0, 60
or 120 mg/kg/day sodium nitrite (0, 12 or 24 mg nitrite-nitrogen/kg/day) by
stomach intubation for 3 days. Mice were sacrificed 11 and 17 days following
treatment and 500 sperm cells per animal from the caudal epididymis were
examined for abnormal morphology. The number of abnormal sperm heads were
elevated in mice given 60 mg/kg/day sodium nitrite, although this elevation
was not statistically significant. Sperm-head abnormalities were
significantly elevated in mice receiving 120 mg/kg/day sodium nitrite. This
t
study identifies a NOAEL of 60 mg/kg/day for sperm-head abnormalities from
sodium nitrite exposure.
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Alavantic ec al. (1988a) investigated the effect of sodium nitrite on
reproduction in mice. Male and female C3H X 101 mice (10-12 weeks old,
25/group) were dosed intragastrically with 0, 60 or 120 mg/kg/day NaN02 (0, 12
or 24 mg nicrite-nitrogen/kg/day) daily for 14 days prior to mating.
Fertility of parent mice, litter size and sex ratio were observed and the
number of heritable translocations and sperm-head abnormalities were assessed
in both parent and offspring. Fertility and litter size were reduced in mice
receiving 60 mg/kg/day, although the reduction was not statistically
significant. A significant reduction in the number of fertile females was
observed at 120 mg/kg/day. Cytogenic evaluation of the spermatocytes of
parental mice revealed a significant elevation of sex chromosome univalency at
120 mg/kg/day NaN02 but not at 60 mg/kg/day. Sodium nitrite did not induce
translocations in stem spermatogonia at diakinesis-metaphase I. Sperm-head
abnormalities were significantly increased at both doses. Nitrite was not
shown to induce heritable chromosomal translocations or sex chromosome
univalency in the offspring of treated mice. No statistically significant
increases in the number of sperm-head abnormalities were apparent in F: mice
and no changes were observed in testes weight or body weight. This study
identifies a LOAEL of 60 mg/kg/day NaN02 for the increased number of spermhead
abnormalities seen in mice.
Roth and Smith (1988) studied the maternally mediated toxicity of nitrite
in Long-Evans hooded rats (200 g) supplied with drinking water containing 0,
2,000 or 3,000 mg NaN02/L prior to and throughout gestation and lactation.
This corresponds to doses of 0, 40 or 60 mg nitrite-nitrogen/kg/day. Pups
from treated dams had reduced growth rates, increased rates of mortality,
severe microcytic anemia, lipemia, fatty liver damage, decreased
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erythropoiesis of boch spleen and bone marrow and decreased plasma and tissue
iron. Treated dams also had significantly reduced plasma iron and reduced
milk iron. Supplementation of the diet with iron reduced the severity of
these effects. This study identifies a LOAEL of 40 mg nitrite-nitrogen/kg/day
for maternally mediated iron deficiency anemia in rat pups.
Shimada (1989) investigated the teratogenic potential of nitrite in mice.
Groups of 16-19 pregnant females were given drinking water containing 0, 100
or 1,000 mg/LUJ from days 7-18 of pregnancy. The average amount of NaN02
consumed was 0, 0.81 or 7.3 mg/mouse/day (about 0, 5.4 or 49 mg nitrite-
nitrogen/kg/day). No detectable changes were noted for maternal weight gain,
pregnancy rate, implants/litter, live fetuses/litters, percentage of dead or
resorbed fetuses/litter, sex ratio, average fetal body weight, number of
runt/litter, type or number of external or skeletal malformations, or
chromosomal aberrations in fetal liver. This study identifies a developmental
NOAEL of 49 mg nitrite-nitrogen/kg/day.
Nitrite plus Nltrosable Substrates
Ivankovic et al. (1973) investigated the teratogenic effect of oral
ethylurea (100 mg/kg) and sodium nitrite (10 mg nitrite-nitrogen/kg) in
pregnant rats. A total of 160 offspring were bom. Four to six weeks after
birth, 96 (60%) of the young animals developed hydrocephalus and died within
2 weeks. No other malformations were seen. Seventeen additional pregnant
(a)The authors reported this a 0, 100 or 1,000 mg/mL. It is presumed that
mg/L was intended.
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Vistar rats were given the sane treatment but vith ascorbic acid (250 mg/kg)
administered orally at the same time. A total of 113 offspring were reared
after birth and none showed symptoms of hydrocephalus 1 year later.
Teramoto et al. (1980) administered ethylenethiourea (400 mg/kg) to
pregnant mice in combination with 50, 100 or 200 mg/kg sodium nitrite (10.2,
20.3 or 40.6 mg/kg as nitrogen). When nitrite was given to mice immediately
after their treatment with ethylenethiourea on day 6 or 9 of pregnancy, fetal
survival was decreased. Various types of malformations were observed in the
living fetuses from dams treated with the two higher doses of nitrite plus
ethylenethiourea on days 6, 8 or 10 of pregnancy, but malformations were not
observed in fetuses from dams treated on day 12. Teratogenicity was not
observed when nitrite was given 2 hours after ethylenethiourea or when either
chemical was administered alone.
D. Mutagenicity
Nitrate
The U.S. Food and Drug Administration (FDA 1972e) evaluated the genotoxic
potential of NaN03 in a number of assays systems. The compound did not
produce a measurable mutagenic response in Salmonella tvphimurium or an
increase in recombination frequency or Saccharomvces cerevislae. either
in vitro or in a host-mediated assay in mice. The compound did not produce
chromosomal abnormalities in bone marrow cells from rats, but did result in a
2-fold increase in the number of acentric fragments in cultures of human
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embryonic lung tissue. No consistent dose-dependent effect was detected in a
dominant lethal gene test in rats. Subsequent tests of KN03 in the dominant
lethal mutation assay were also negative (FDA 1979a).
Ishidate et al. (1984) studied the effect of NaN03 on the reverse
mutation rate in Salmonella tvphimurium strains TA92, TA1535, TA100, TA1537,
TA94 and TA98, with or without activation by rat liver microsomal fractions
(S-9). Chromosome aberration tests In vitro were carried out using a cultured
Chinese hamster fibroblast (CHL) cell line without metabolic activation.
Sodium nitrate did not induce a significant increase in the number of
revertant colonies in the Salmonella/microsome assay, but did increase the
frequency of chromosomal aberrations in CHL cells.
Luca et al. (198S) administered 2 gavage doses of sodium nitrate at
24-hour intervals to male Wistar rats (10-12 weeks old). The doses
administered were 0, 78.5, 235.5, 706.6 or 2,120 mg NaN03/kg (0, 16, 47, 140
or 420 mg nitrate-nitrogen/kg). Rats were sacrificed 24 hours after the
second dose was administered. Chromosomal aberrations (gaps, breaks, acentric
fragments and exchanges) were examined in metaphase cells from bone marrow.
No significant differences in the number of structurally abnormal chromosomes
were seen in treated rats. When exposure was extended to 2 weeks, the
percentage of aberrant metaphases was significantly increased in treated rats
in an apparent dose-related fashion.
Luca et al. (1985) administered 2 gavage doses (at 24 hour intervals) of
sodium nitrate to groups of 8 male Swiss mice (12-14 weeks old). The doses
administered were 0, 78.5, 235.5, 706.6 or 2,120 mg NaN03/kg (0, 13, 39, 120
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or 350 mg nitrate-nitrogen/kg). Mice were sacrificed 6 hours after the second
dose for the evaluation of micronuclei or 24 hours after the second dose for
examination of chromosome aberrations (gaps, breaks, acentric fragments and
exchanges) in metaphase cells from bone marrow. The percent of cells with
structurally abnormal chromosomes was not significantly affected by treatment
except in the group of mice receiving 706.6 mg/kg NaNOj. An effect was not
seen in mice receiving the highest dose.
Alavantic et al. (1988b) studied the effect of NaN03 on the rate of
unscheduled DNA synthesis (UDS) in spermatids in male mice. Groups of
5 animals (10-12 weeks old) were exposed to either 0, 600 or 1,200 mg/kg/day
sodium nitrate by stomach intubation (0, 100 or 200 mg nitrate-
nitrogen/kg/day). Mean levels of UDS measured by injection of 3H-thymidine
were not significantly affected at either dose level.
Nitrite
The U.S. Food and Drug Administration (FDA 1972f) evaluated the genotoxic
potential of NaN02 in a number of assays systems. The compound did not
produce a measurable mutagenic response in Salmonella tvphimuriura or an
increase in recombination frequency in Saccharomvces cerevisiae in a
host-mediated assay in mice, but did yield positive responses in both
organisms in vitro. The compound did not produce chromosomal abnormalities in
bone marrow cells from rats, but did result in a sharp increase in the number
of acentric fragments in cultures of human embryonic lung tissue. No
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consistent dose-dependent effect was detected in a dominant lethal gene test
in rats. Subsequent tests of KN02 in the dominant lethal mutation assay were
also negative (FDA 1979b).
Couch and Friedman (1975) found no evidence of mutagenicity for sodium
nitrite in a host-mediated assay in male ICR mice. Salmonella tvphimurium
were injected intraperitoneally immediately after gavage with 150 mg/kg sodium
nitrite (30 mg/kg nitrite-nitrogen).
Kodama et al. (1976) studied the mutagenic effect of sodium nitrite on a
cultured mouse mammary tumor cell line. Severe chromosomal aberrations were
induced in 40% of the cells incubated with 3 mM and 80% at 10 mM NaN02. The
frequency of 8-azaguanine resistant cells was increased in cultured incubated
with concentrations greater than 1 mM NaN03.
Whong et al. (1979) injected Salmonella tvphimurium intravenously into
female mice and rats, followed by administration of NaN02 by gavage ten
minutes later. The dose was 160 mg/kg (32 mg/kg nitrite-nitrogen) for mice
and 150 mg/kg (30 mg/kg nitrite-nitrogen) for rats. No evidence of increased
mutation frequency in the bacteria was detected.
Inui et al. (1980) reported a small increase (3.7 times control) in to
8-azaguanine resistance in Syrian hamster embryo cells cultured 24 hours after
gavage of pregnant hamsters with 100 mg/kg sodium nitrite (20 mg/kg nitrite-
nitrogen) .
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El-Nahas et al. (1984) studied the mutagenic potential of sodium nitrite
on adult bone marrow and embryonic liver cells of the rat. Drinking water
containing 1,250 mg sodium nitrite/L was provided to pregnant albino rats on
days 5 through 18 of gestation. The authors estimated that this resulted in
an average sodium nitrite dose of 42 mg nitrite-nitrogen/kg/day. Pregnant
rats were sacrificed and 30 treated and 10 control embryonic livers retrieved.
Bone marrow was obtained from the femurs of 6 adult control and 7 adult
nitrite-treated rats. Adult bone marrow metaphase and metaphase cells from
embryonic livers were examined for chromosomal aberrations. The number of
chromosomal aberrations (breaks, centric fusions, dicentrics) were
significantly increased in both adult bone marrow and embryonic liver.
Chromosomal breaks were the most frequently seen, occurring in 7.3% of
examined maternal bone marrow cells from treated rats compared to 3.3% of
controls and in 6.4% of embryonic liver cells examined compared to 1.7% of
controls.
Ishidate et al. (1984) reported that NaN02 produced a clear dose-
dependent increase in mutation frequency in Salmonella typhimurium. with or
without metabolic activation. Incubation of Chinese hamster fibroblast cells
with NaK02 (250, 500 or 1,000 mg/L) la vitro resulted in a dose dependent
increase in the incidence of chromosomal structural aberrations (11%, 16% and
22%, respectively).
Luca et al. (1987) administered sodium nitrite by gavage to groups of
6-8 male mice or rats at doses of 0, 1.72, 5.18, 15.55 or 46.66 mg/kg in
distilled water (0.34, 1.0, 3.1 or 9.3 mg/kg nitrite-nitrogen). Animals were
given 2 doses at 24 hour intervals and sacrificed 24 hours after the second
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nicrite dose. Bone marrow was examined for chromosomal aberrations (gaps,
breaks, acentric fragments and exchanges) (200 cells scored per group) and
micronuclei analysis performed. The percent of aberrant metaphases were
significantly increased in treated mice and rats but there was no apparent
dose-related trend. Micronucleated polychromatic erythrocytes (studied in
mice only) were significantly increased in all but the high-dose group. The
authors interpreted this to suggest that NaN02 was mutagenic at doses below
its cytotoxic level.
Luca et al. (1987) administered 0, 1.72, 5.18, 15.55 or 46.66 mg/kg
sodium nitrite in drinking water daily to groups of 6 male rabbits
(30-32 weeks of age) for 3 months. Rabbits were sacrificed 24 hours after the
last dose of sodium nitrite. Bone marrow was examined for chromosomal
aberrations (gaps, breaks, acentric fragments and exchanges). Structural
aberrations were significantly increased in the two low dose groups only.
Luca et al. (1987) incubated cultured monkey liver cells (BS-C-1) and
HeLa cells with sodium nitrite in physiological saline at concentrations of
0.265 mg/mL or 0.530 mg/mL (0.053 or 0.106 mg nitrite-nitrogen/mL) . Both
doses resulted in significantly increased percentages of aberrant metaphases
in the cultured cells, but no dose-related trend was observed.
Mukherjee et al. (1988) studied the effect of sodium nitrite on sister
chromatid exchange (SCE) and micronuclei (MN) in mice. All animals were
implanted with a paraffin-coated tablet containing 5-bromo-deoxyuridine.
Sodium nitrite was administered by a single intraperitoneal injection of 25,
50, 75, 100 or 150 mg/kg (5, 10, 15, 20 or 30 mg nitrite-nitrogen/kg), and
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animals were sacrificed 24 or 48 hours later. All doses of nitrite resulted
in statistically significant increases in the incidence of SCE and MN in bone
marrow cells. Treatment with sorbic acid did not reduce these effects.
Alavantic et al. (1988b) studied the effect of sodium nitrite on the rate
of unscheduled DNA synthesis (UDA) in spermatids in male mice. Groups of
5 animals (10-12 weeks old) were exposed to 0, 60 or 120 mg sodium
nitrite/kg/day (12 or 24 mg nitrite-nitrogen/kg/day). Mice were sacrificed
17 days after treatment for evaluation of UDS. Mean levels of UDS (measured
by injection of 3H-thymidine) were not significantly different from controls
at either dose level.
Nitrite plus substrate
Couch and Friedman (1975), Whong et al. (1979) and Inui et al. (1980)
reported significant mutagenicity in stomach juice of animals following gavage
with various nitrosatable substrates and sodium nitrite. These results are
possibly due to the formation of mutagenic N-nitroso compounds in the stomachs
of these rodents, rather than direct mutagenicity of sodium nitrate. Two
other studies in mice detected mutagenic activity after gavage of nitrite and
five other nitrosatable substrates using Salmonella tvphimurlua injected
intraperitoneally or intravenously as genetic indicator (Braun et al. 1977,
1980).
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E. Carcinogenicity
Effects of Nitrate Alone
Greenblatt and Mirvish (1972) supplied groups of 40 strain A mice with
sodium nitrate in water at concentrations of 0 or 12,300 mg/L. This
corresponds to doses of about 0 or 340 mg nitrate-nitrogen/kg/day. The
animals were exposed for 25 weeks, and the lungs of the animals were examined
histologically after an additional 13 weeks. The numbers of lung tumors in
the treated and control mice were similar. No other tissues were examined.
The treatment period may have been too short for carcinogenic effects to have
occurred.
Lijinsky et al. (1973) administered 100 mg of sodium nitrate daily in
drinking water to each of 15 male and 15 female MRC rats. This corresponded
to a dose of 39 mg nitrite-nitrogen/kg/day. Administration was 5 days a week
for 84 weeks followed by a 20-week observation period. An increase in
pituitary tumors was observed in treated females (11/15 treated vs 3/15
controls). The authors stated "The high incidence of tumors of normal type in
both experimental and control groups is unexpected and difficult to explain...
Our groups of animals were small, and statistical analysis of the results
could not be conclusive".
Maekawa et al. (1982) performed a 2-year bioassay of the carcinogenicity
of sodium nitrate in F344 rats. Groups of 50 males and 50 females were
administered 0, 25,000 or 50,000 ppm NaN03 in the diet. This corresponds to
doses of 0, 200 or 400 mg nitrate-nitrogen/kg/day. Tumor incidence in all
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groups was close to 100%, but there were no significant differences in mean
survival time, mean time to tumor, incidence of malignant tumors or incidence
of any specific tumors.
Effect of Nitrite Alone
Garcia and Lijinsky (1973) administered sodium nitrite in drinking water
at concentrations of 0 or 200 mg/L (corresponding to doses of about 0 or
5.5 mg nitrite-nitrogen/kg/day) to 15 MRC rats of each sex for 75 weeks.
Animals were kept until death and subjected to complete pathological
examination. There were a total of 19 tumor-bearing animals in the treated
groups compared to nine in the controls. The only notable specific increase
was seven pituitary tumors in treated rats compared to three in controls.
This doubling was not statistically significant.
Sen et al. (1975) maintained groups of 20 male English short-hair guinea
pigs with drinking water containing 0 or 800 mg/L sodium nitrite (160 mg
nitrite-nitrogen/L) for 30 months. Although all guinea pigs were subjected to
gross examination and histopathologic examination of nine major organs, only
the observation regarding liver tumors were reported. There were no liver
tumors in the unexposed or nitrite-exposed guinea pigs.
Shank and Newberne (1976) exposed 96 Sprague-Dawley rats of both sexes to
sodium nitrite in the diet at a level of 0 or 1,000 ppm for 125 weeks. This
corresponds to doses of 0 or 10 mg nitrite-nitrogen/kg/day. Exposed animals
had a 27% incidence of tumors of the lymphoreticular system. This was
compared to a 6% incidence in 156 untreated control rats. Significant
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increases in tumor incidence were not observed in other tissues. No tumors of
the lymphoreticular system or any other carcinogenic effect were found in
Syrian golden hamsters treated similarly.
In a follow-up study, Newberne (1978, 1979) administered sodium nitrite
to groups of 68 Sprague-Dawley rats of each sex in water or in various diets
from the time of conception through their natural life span. Doses ranged
from 12.5 to 200 mg/kg/day (4-60 mg nitrite-nitrogen/kg/day). The rats
survived well. The author observed the usual spectrum of neoplastic and
non-neoplastic diseases associated with old rats. The tissue changes of most
interest were observed in the spleen, lymph nodes and other components of the
lymphoreticular tissues. The spleen exhibited a lesion described to as
immunoblastic cell proliferation. When spleen and non-spleen lymphomas were
combined, there were suggestions of a nitrite effect but there was no
convincing dose-response relationship. Combining all nitrite-treated groups
and all control groups, the incidence rates for lymphomas were 8.5% and 12.5%
in control and nitrite - treated groups, respectively. Overall, the incidence
rates of lymphoreticular neoplasia were 15.3% in untreated and 23.7% in
nitrite-treated animals. Newberne's histopathologic assessment of the tissues
indicated that, by considering all the groups receiving sodium nitrite
together, there was a statistically significant excess of lymphoid tumors
(P<0.01 based on chi-square analysis). This was reflected especially in the
groups receiving sodium nitrite in drinking water.
After Newberne's report was submitted, a Government Interagency Working
Group on Nitrite Research reviewed a sample of histological slides from the
study and decided that there was sufficient difference of opinion in the
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diagnoses to warrant a further evaluation of the histopathological findings.
The Universities Associated for Research and Education in Pathology (UAREP), a
nonprofit consortium of 15 universities organized to carry out educational and
research activities in pathology, was selected by the FDA to review the slides
(FDA 1980a). A Joint Committee of Experts was established by the UAREP to
perform this review. The committee members diagnosed fewer lymphomas than
Newberne had reported. The disparity between the two series of diagnoses
involved the differentiation of lymphomas from extramedullar^ hematopoiesis,
plasmacytosis or histiocytic sarcoma. Furthermore, the committee did not
interpret the lesions in the spleen and lymph nodes as immunoblastic
hyperplasia as did Newberne. These lesions were interpreted predominantly as
extramedullary hematopoiesis and plasmacytosis in the spleen and plasmacytosis
or reactive lymphoid hyperplasia in lymph nodes.
In its final report to the FDA, the Government Interagency Working Group
summarized the UAREP committee's findings and concluded that most of the
lymphoma diagnoses originally reported were not confirmed. The UAREP
pathologists diagnosed fewer lesions as lymphoma, with a resulting reduction
of incidence to approximately 1% among treated and control groups. This rate
of lymphoma incidence is similar to that usually seen spontaneously in
Sprague-Dawley rats. UAREP pathologists did report a greater than 1%
incidence of other types of tumors, including histiocytic sarcomas. However,
no demonstration could be found that the increased incidences of these tumors
were induced by the ingestion of sodium nitrite (FDA 1980b).
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Taylor and Li jinsky (1975) and Lijinsky and Taylor (1977) provided
drinking water containing 2,000 mg/L sodium nitrite (400 mg nitrite-
nitrogen/L) to groups of 27 male and 30 female Sprague-Dawley rats 5 days/week
for 104 weeks. Untreated controls were not maintained, but based on complete
gross and microscopic pathologic examination of the nitrite-exposed group,
Lijinsky and Taylor (1977) concluded that the identity and incidence of tumors
observed were those expected in aged rats of this strain.
Inai et al. (1979) supplied sodium nitrite at concentrations of 1,250,
2,500 or 5,000 mg/L in drinking water (about 42, 83 or 167 mg nitrite-
nitrogen/kg/day) to groups of 50 male and 50 female ICR mice for 18 months.
No tumors attributable to nitrite treatment were observed in histopathological
examinations of major organs.
Anderson et al. (1979) provided drinking water containing 14 mM sodium
nitrite (200 mg nitrite-nitrogen/L) 4 days/week to 20 female and 17 male
weanling Swiss mice for 8 months. Another group of 16 female and 21 male mice
served as untreated controls. The mice were observed until spontaneous death,
at which time they were subjected to necropsy and histopathological
examination of all gross lesions. There was no evidence of a carcinogenic
effect of exposure to nitrite.
Mirvish et al. (1980) reported that papillomas of the forestomach
developed in 8 of 45 (18%) MRC Vistar rats (both sexes) administered an
aqueous solution containing 3,000 mg/L of sodium nitrite (57.7 mg/kg/day as
nitrogen) 5 days/week for life. Two of 91 untreated rats (2%) developed these
tumors (P<0.002). These rats were fed Wayne Lab-Blox which contains fish
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meal, a source of nitrosatable amines, so this effect might have been due Co
formation of nitrosamines rather than nitrite per se (see below).
Pearson et al. (1980) administered drinking water containing 0 or
1,000 mg/L sodium nitrite (200 mg nitrite-nitrogen/L) to groups of 6 weanling
mice (strain and sex not specified), maintained on a complete laboratory chow
diet for 12 months. Survivors were killed at 12 months and subjected to gross
examination and histopathologic examination of the liver, lungs, heart, spleen
and stomach. This experiment was repeated 3 times over the next 3 years,
using 10 mice per group. Thus, a total of 36 mice/group was examined. The
tumor incidence in the nitrite-created group was not significantly different
from that in the nonexposed group.
Maekawa et al. (1982) supplied groups of 50 male and 50 female F344 rats
with water containing 0, 1,250 or 2,500 mg/L of NaN02. This corresponds to
doses of about 0, 2.5 or 5 mg nitrite-nitrogen/kg/day. Exposure was for
2 years. Tumor incidence in all groups was close to 100%, but there were no
significant differences in mean survival time, mean time to tumor, increase of
malignant tumors or increase of any specific tumor.
Lijinsky et al. (1983) provided two groups of 24 male and two groups of
24 female F344 rats (7-8 weeks old) with free access to food containing
2,000 ppa sodium nitrite (20 mg/kg/day as nitrite-nitrogen). One group of
24 females and one group of 24 males were given access to 2,000 mg/L sodium
nitrite in their drinking water (56 mg/kg/day nitrite nitrogen), 5 days per
week for 2 years. Untreated controls were included for each sex. Survival
rates of treated rats were not significantly different from controls. Female
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rats exposed to nitrite in food had significantly higher incidences of
hepatocellular neoplasms (27/48 vs 8/48, P-0.0001), although the number of
liver carcinomas by themselves was not statistically different. No
significant differences in the incidence of liver neoplasms were seen in
males. The incidence of mononuclear cell leukemia was significantly reduced
in nitrite-fed rats (males and females) and in female rats receiving nitrite
in their drinking water.
In a two-generation carcinogenicity study, Olsen et al. (1984) fed a diet
containing potassium nitrite-treated meat to 70 male and 140 female Wistar
rats. The resultant nitrite content of the diet was reported to be 2, 4 or
94 mg/kg. This corresponds to doses of 0.02, 0.04 or 0.9 mg nitrite-
nitrogen/kg/day. Rats were fed this diet for 10 weeks prior to mating. Male
and female offspring (60-100/group) were continued on the same diet after
weaning. At 128 weeks, all surviving F1 rats were sacrificed and histological
examination was conducted on an extensive group of tissues. The number of
malignant tumors found in treated rats was not significantly greater than in
controls, but there was a trend for increased dermal squamous cell carcinomas
in males. A statistically significant increase in males with kidney adenomas
was observed, but this could have been by chance. The doses used in this
study are too low to draw reliable conclusions about the effects of nitrite on
cancer incidence.
Ernst et al. (1987) supplied groups of 15 male and 15 female Syrian
hamsters with food containing sodium nitrite at 0 or 2,000 ppm (400 mg
nitrite-nitrogen/kg food), 5 days/week for their lifetimes. At death, the
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hamsters were subjected to a comprehensive gross and histopathologic
examination. There were no significant differences between untreated and
nitrite-exposed groups in the incidence of tumors.
Grant and Butler (1989) studied the carcinogenic effect of lifetime
exposure of rats to nitrite. Groups of 50 males were given diets containing
0, 0.2% or 0.5% NaN02 for 115 weeks. This corresponds to average doses of 0,
20 or 50 mg nitrite-nitrogen/kg/day. Thorough histological examinations
revealed no statistically significant increase in tumor incidence in any
tissue. Significant decreases in tumor incidence were noted for total
lymphomas, mononuclear cell leukemia and testicular interstitial cell tumors.
Lijinsky and Kovatch (1989) provided drinking water containing 0 or 0.2%
sodium nitrite to F344 rats (8-weeks old, 19-24/group), 5 days per week for at
least 2 years. This corresponds to doses of 0 or 40 mg nitrite-
nitrogen/kg/day. No adverse effects (including increased mortality) were seen
in treated rats, and the number and type of neoplasms were not significantly
affected by treatment. The incidence of mononuclear cell leukemia was
significantly decreased.
Effect of Nitrite Administered with Nitrosable Substrates
Greenblatt et al. (1971) provided groups of 40 male and 40 female mice
with water containing at 1,000 mg/L of NaN02 (5 days/week) with or without
piperazine (6,250 mg/kg), morpholine (6,330 mg/kg) or methylaniline
(1,950 mg/kg) in the diet. Exposure was terminated after 28 weeks. Animals
were sacrificed after an additional 12 weeks, and the lungs were examined for
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Che presence of tumors. Exposure to NaN02 in combination with any one of the
amines resulted in a statistically significant increase in both the percentage
of animals with tumors and the number of tumors per tumor-bearing mouse. This
was more pronounced in males than females. Exposure to NaNOz or the amines
alone did not cause increase in the incidence of tumors. The authors
concluded that tumor induction was due to in vivo formation of carcinogenic
nitrosamines.
Mirvish et al. (1972) supplied groups of 31-74 Swiss mice with food
containing methylurea or ethylurea (5,360 ppm or 6,360 ppm, respectively)
along with water containing 0 or 1,000 mg/L of NaNOz. This corresponds to a
dose of 0 or 30 mg nitrite-nitrogen/kg/day. Animals were exposed for 20 weeks
and sacrificed 12 weeks later. Exposure to alkylurea plus nitrite resulted in
statistically significant increases in the percentage of mice with lung
adenomas and in the number of adenomas per mouse. Neither alkylurea nor NaN02
alone had a significant effect on tumor incidence.
Greenblatt and Mirvish (1972) supplied groups of 40 male mice with diets
containing 0 or 6,250 ppm of piperazine, along with water containing 0, 50,
250, 500, 1,000 or 2,000 mg/L NaN02. This corresponds to a dose of about 0,
1.5, 7.5, 15, 30 or 60 mg nitrite-nitrogen/kg/day. Exposure was for 20 weeks.
The animals were sacrificed and examined for lung tumors 10 weeks later.
There was a clear dose-response trend toward increased numbers of mice with
adenomas and adenomas per adenoma-bearing mouse (P<0.001 for all doses except
50 mg/L). Exposure to piperazine alone (6,250 ppm) or nitrite alone
(2,000 mg/L) did not result in a significant increase in tumors.
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Greenblact et al. (1973) exposed groups of rats (15 males plus 15
females/group) to NaN02 and one of three secondary amino acids (proline,
hydroxyproline, arginine) in drinking water. Doses were 10 mg nitrite-
nitrogen/kg/day plus 25 mg/kg/day of amino acid. After 122 weeks, animals
were sacrificed and subjected to histological examination for tumors. There
were no detectable increases in the incidence of any tumors in any exposure
group. The authors concluded that the nitrosoamino acids presumably formed by
this treatment were not carcinogenic.
Taylor and Lijinsky (1975) administered 0.2% sodium nitrite (27 mg
nitrite-nitrogen/kg/day) and 0.2% heptamethyleneimine (133 mg/kg/day) in
drinking water to 15 male and 15 female Sprague-Dawley rats fed Purina lab
chow. Rats were 8- to 10-weeks-old at the start and were treated for 28 weeks
and observed until death. This treatment induced tumors of the oropharynx,
tongue, forestomach or esophagus in 27 of 30 rats. No tumors were observed in
animals exposed to nitrite alone or heptamethyleneimine alone.
Mirvish et al. (1976) tested the effect of sodium ascorbate on the
oncogenic response induced by a combination of sodium nitrite and morpholine
in groups of 40 eight-week-old male MRC-Wistar rats. In this study, three
groups were used. Group I was treated with 3,000 mg/L of sodium nitrite in
drinking water (81 mg nitrite-nitrogen/kg/day) and 500 mg/kg/day morpholine in
food. Group II was administered the same treatment plus 1,135 mg/kg/day
sodium ascorbate in food. The control group was kept on normal diet and plain
water. Liver tumors appeared in 65% of group I, 49% of group II and none of
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the controls. Forestomach tumors appeared in none of group I, 54% of group II
and 4% of untreated animals. Thus, in this study, a marked increase in
forestomach tumors was observed in animals that received sodium ascorbate.
Lijinsky and Rueber (1980) administered 0 or 0.2% sodium nitrite and 0 or
0.1% disulfiram in a powdered diet to 40 Fischer 344 rats. This corresponds
to doses of 0 or 20 mg nitrite-nitrogen/kg/day. Rats were 8- to 9-weeks-old
at the start of treatment which continued for 78 weeks followed by observation
until natural death. Exposure to nitrite plus disulfiram induced tumors of
the stomach, esophagus, tongue and nasal cavity in 10 of 20 male and 12 of 20
female rats. None of these tumors were found in rats administered nitrate or
disulfiram alone. The nitrosation product of disulfiram is
nitrosodiethylamine.
Bergman and Wahlin (1981) supplied groups of 10 Syrian hamsters with
water containing 0 or 0.1% aminopyrine and 0 or 0.1% NaN02. This corresponds
to a dose of 0 or 17 mg nitrite-nitrogen/kg/day. After 20 weeks, animals were
sacrificed and examined for tumors of the liver, gallbladder, lungs and
kidneys. No tumors were observed in animals supplied with aminopyrine or
nitrite alone. Nearly all animals in the group exposed to both aminopyrine
and nitrite showed multiple foci of bile duct adenomas, and 6/10 developed
cholangiocarcinoma of the liver. No tumors were detected in other tissues.
Lijinsky (1984) exposed groups of 20-24 male rats to 0.2% NaN02 in
drinking water or food for their lifetimes. This corresponds to doses of
about 20-40 mg nitrite-nitrogen/kg/day. Animals were also exposed to one of
four nitrosable amines (allantoin, dimethyldodecylamine-N-oxide,
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diphenhydramine and chlorpheniramine) in the diet or in drinking water
(0.1%-0.2%). None of the amines along resulted in increased incidence of
liver neoplasms, but NaN02 plus diphenhydramine, chlorpheniramine or
dimethyldodecylamine oxide all resulted in significant increases in liver
tumors (hepatocellular carcinomas and neoplastic modules). A marginal
increase was noted for allantoin plus NaN02.
Thanavit et al. (1988) administered aminopyrine (0 or 1,000 mg/L) and
NaN02 (0 or 1,000 mg/L) in drinking water to groups of 14-18 male Syrian
hamsters for 8-10 weeks. This corresponds to doses of 0 or 20 mg nitrite-
nitrogen/kg/day. After an additional period of 2-20 weeks, the animals were
sacrificed and subjected to histological examination for liver neoplasms. No
tumors or nodules were detected in control animals or in animals exposed to
nitrate or aminopyrine alone. In animals exposed to both, 3/17 animals had
liver carcinomas, 7/17 animals has cholangiofibrosis and 2/17 had
hepatocellular nodules. The incidence of these tumors was further increased
by prior dosing with liver parasites that caused inflammatory and
proliferative responses in the liver.
Mokhtar et al. (1988) exposed groups of 80 male mice to water containing
2,000 mg/L NaN02 and 1,000 ppm dibutylamine. This corresponds to a dose of
40 mg nitrite-nitrogen/kg/day. Histopathological examinations of liver were
performed on groups of 15-20 after 3, 6, 9 or 12 months of exposure. Exposed
animals developed hepatic dysplasia and necrosis within 3-6 months, and this
became more pronounced with continued exposure. Hepatomas were found in
3/20 mice exposed to 9 months and 1/15 mice exposed to 12 months. No liver
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tumors were detected in control mice (exposed to neither NaN02 nor
dibutylamine) of the same age. This tumorigenic response was diminished by
concurrent feeding of soybean diet or ascorbic acid.
Yamamoto et al. (1989) administered bis(2-hydroxylpropyl)amine (BHPA) in
the diet to groups to 20 male rats, along with drinking water containing 0,
1,500 or 3,000 mg/L of NaN02. This corresponds to doses of 0, 30 or 60 mg
nitrite-nitrogen/kg/day. Exposure was for 94 weeks. Histological examination
of tissues revealed an increased incidence of tumors of the nasal cavity
(14/19), lung (11/19) and esophagus (2/19) in the high dose group. In the low
dose group, increased tumors were seen only in lung (3/19). No tumors were
seen in any of these tissues in control animals or in animals exposed to BHPA
or NaN02 alone. The authors attributed this effect to formation of
N-nitrosobis(2-hydroxypropyl)amine (BHP) in vivo. This was supported by the
detection of BHP in the urine of animals exposed to BHPA and NaN03, but not in
other groups.
Studies on In Vivo Nltrosamine Formation
Inui et al. (1980) administered 25, 50 or 100 mg/kg aminopyrine and 25,
50 or 100 mg/kg sodium nitrite (5, 10 or 20 mg nitrite-nitrogen/kg) to adult
golden hamsters by gavage. Stomach contents were then examined for the
presence of nitrosamines. A dose-dependent increase in N-nitrosodimethylamine
(0.1-1.49 mg/animal) was measured within 30 minutes of the administration of
aminopyrine and sodium nitrite. The concentration of dimethylnitrosamine in
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the stomach was much lower (0.01-0.04 mg/animal) in hamsters treated with
sodium nitrite alone, and was not detectable in animals treated with
aminopyrine alone or in untreated controls.
Massey et al. (1988) studied endogenous nitroso compound formation in
normal and germ-free rats supplied with nitrite in drinking water (0, 50 or
500 mg N02/L) . Nitroso compounds could not be detected in blood or tissues of
any group, but were detected in the stomach and intestines of conventional
rats at levels that increased as a function of nitrite concentration in water.
Nitroso compounds were detected in stomach and intestines of germ-free rats,
but at levels lower than conventional animals. These data were interpreted by
the authors to indicate that enteric bacteria converted dietary or endogenous
substrates to nitroso compounds, and that increased levels of nitrite in water
increased the amount of nitroso compound formation.
F. Summary
The toxic effects of nitrate are closely related to its conversion to
nitrite by bacteria in the alimentary tract. Thus, the toxicity of nitrate
depends not only on dose, but also on the level and type of bacteria present.
Consequently, dose-response relationships are quite variable between different
animal species. For example, ruminants are highly sensitive, while dogs have
low sensitivity. Because of this species variability, animal data should be
extrapolated to humans with caution.
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The principal health effect from exposure to nitrate or nitrite is
methemoglobinemia. This results when nitrite oxidizes the Fe+2 form of iron
in hemoglobin to the Fe+3 state. In rats, acute oral LD50 values for NaN03
range from 1,000 to 2,000 mg/kg. These are close to LD}0 values for NaCl,
suggesting that acute lethality from NaN03 is caused mainly by nonspecific
electrolyte imbalance. Chronic exposure to doses up to 69 mg nitrate-
nitrogen/kg/day did not produce methemoglobinemia in rats, but doses of
50 mg/kg/day or higher have been noted to produce mild histological changes in
liver or spleen. High doses nay also interfere with thyroid function.
Nitrite is substantially more toxic to rodents than nitrate. Acute oral
LDJ0 values range from 40 to 120 mg/kg (20-40 mg nitrite-nitrogen/kg/day),
while doses of 20 mg nitrite-nitrogen/kg/day can produce methemoglobinemia and
other related histological effects. High doses of nitrite act on vascular
smooth muscle to produce vasodilation and hypotension, and may also cause
changes in coronary blood vessels.
Numerous studies on reproductive function, teratogenicity and fetal
development in several animal species have not revealed evidence of adverse
effects from nitrate or nitrite except at relatively high dose levels. Such
effects are likely to be secondary to maternal or fetal methemoglobinemia and
related toxicity. Exposure of males to high doses may result in cytogenetic
abnormalities in spermatocytes and morphological abnormalities in sperm, but
this did not result in any apparent effect on reproduction or offspring.
Ingestion of 20-100 mg nitrite-nitrogen/kg/day by dams during lactation can
cause iron deficiency in milk and moderate to severe anemia and other post-
natal effects in sucklings.
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Most investigations on the mutagenicity of nitrate and nitrite have been
negative. However, many studies have reported that both nitrate and nitrite
lead to a variety of chromosomal aberrations, both in cultured cells in vitro
and in animals exposed in vivo.
The carcinogenic potential of nitrate has not been extensively studied in
animals, but several investigations have not revealed any evidence of
increased tumor incidence. The carcinogenic potential of nitrite has been
much more thoroughly studied. One early study was interpreted as providing
evidence of increased tumors of the lynphoreticular system in rats, but
re-evaluation of the histological slides from this study led to the conclusion
that the lesions were not neoplastic. Numerous other studies of nitrite
carcinogenicity in animals have also been negative. However, when nitrite is
given to animals along with high levels of nitrosable amines, clear increases
in tumor incidence are often observed in a variety of tissues. This
tumorigenic response is probably a result of the formation of carcinogenic
nitrosamines by reaction of nitrite with the nitrosable substrates in the
stomach.
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VI. HEALTH EFFECTS IN HUMANS
A. Methemoglobinemia
The principal adverse health effect associated with acute exposure of,
humans to nitrate or nitrite is methemoglobinemia. Low levels of
methemoglobin occur in normal individuals, with typical values usually ranging
from 0.5% to 3.0% (Winton et al. 1971, ECETOC 1988). However, due to the
large excess capacity of blood to carry oxygen, levels of methemoglobin up co
around 10% are not associated with any significant clinical signs (Walton
1951, Winton et al. 1971, ECETOC 1988), Concentrations above 10% may cause a
bluish-color to skin and lips (cyanosis), while values above 25% lead to
weakness, rapid pulse and tachypnea (Jones et al. 1973). Death may occur if
methemoglobin values exceed 50%-60%.
Nitrite converts hemoglobin to methemoglobin by oxidizing the Fe+2 ion in
heme to the Fe+3 state. Nitrate does not carry out this reaction unless it is
first converted to nitrite. Conversion of nitrate to nitrite is mostly
mediated by bacteria in the gastrointestinal system (mouth, stomach).
Consequently, the risk of methemoglobinemia from ingestion of nitrate depends
not only on the dose of nitrate, but also on the number and type of enteric
bacteria. In adults, available data suggest about 5% of a dose of nitrate is
reduced to nitrite (ECETOC 1988). Infants are more susceptible to nitrate
toxicity than adults, since the high pH of the infant gastrointestinal system
favors the growth of nitrate-reducing bacteria. In particular, infants
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exposed to bacteriologically-contaminated waters are susceptible to
methemoglobinemia, since the ingested bacteria are likely to flourish in the
stomach (ECETOC 1988) .
1. Methemoglobinemia in Infants
Comblath and Hartmann (1948) supplied water containing 1,000 mg N03/L
to four infants (ages 11 days to 11 months) for 2-18 days. The amount
administered yielded doses of 50 mg N03/kg/day (11 mg nitrate-
nitrogen/kg/day) . This intake resulted in a methemoglobin concentration of
5.3%, but there was no evidence of cyanosis. Four additional infants (2 days
to 6 months of age) were given 100 mg N03/kg/day (23 mg nitrate-
nitrogen/kg/day) for periods of 6-9 days. No cyanosis was evident in these
infants, and the highest methemoglobin concentration observed was 7.5% (at
8 days in a 10-day-old infant). This study identifies a N0AEL of 23 mg
nitrate-nitrogen/kg/day, which corresponds to 140 mg nitrate-nitrogen/L in
water (assuming intake of 0.16 L/kg/day by an infant).
Donahoe (1949) reported five cases of moderate to severe cyanosis in
infants in South Dakota. In four of the five cases, the water used to feed
the infants vas from shallow wells and was shown to be heavily contaminated
with bacteria. Nitrate levels were measured in two cases, with values of 50
and 177 mg/L (12 and 41 mg nitrate-nitrogen/L), respectively. This
corresponds to doses of 8 and 28 mg nitrate-nitrogen/kg/day.
Bosch et al. (1950) evaluated 139 cases of methemoglobinemia reported by
physicians in Minnesota. All of the cases were in young children, with 90%
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occurring in infants less than 2 months of age. A study of the nitrate .
concentration of the wells (a total of 129) used to supply water to the
children with methemoglobinemia was performed. None of the wells contained
less than 10 mg/L nitrate-nitrogen. Two wells (1.5%) contained 10-20 mg/L,
although the diagnosis of methemoglobinemia was considered questionable in
both these cases. There were 25 wells (19%) that contained 21-50 mg/L,
53 (41%) that contained 51-100 mg/L, and 49 (38%) that contained more than
100 mg/L nitrate-nitrogen. Nearly all the wells were shallow dug wells with
inadequate protection from surface contamination. Coliforii organisms were
detected in 45 of 51 samples (88%) of samples tested for bacterial
contamination.
Walton (1951) presented the results of a survey conducted by the American
Public Health Association on morbidity and mortality among infants due to
methemoglobinemia resulting from the ingestion of nitrate contaminated water.
The survey was conducted by means of a questionnaire sent to all 50 states.
The results of the survey revealed over 278 cases of infant methemoglobinemia,
including 39 deaths, which were shown to be "definitely associated with
nitrate-contaminated water." These occurred in 14 different states, located
mostly in the north-central portion of the U.S. Of 214 cases for which
quantitative data were available on nitrate levels in water, none occurred in
infants consuming water containing less than 10 mg nitrate-nitrogen/L. There
were 5 cases (2%) in infants exposed to 11-20 mg nitrate-nitrogen/L, 36 cases
(17%) in infants exposed to 21-50 mg/L and 173 (81%) in infants exposed to
more than 50 mg/L. Data on levels of bacteriological contamination, incidence
of gastrointestinal disease, nitrite levels or actual methemoglobin levels
were not reported.
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Eving and Hayon-White (1951) reported two cases of severe
methemoglobinemia in infants. In the first, the infant ingested water from a
shallow well that was "heavily polluted" with bacteria and contained 200 mg/L
of nitrite (46 mg nitrate-nitrogen/L, equivalent to a dose of 7.4 mg nitrate-
nitrogen/kg/day). This infant died. In the second case, the infant ingested
water from a shallow well heavily contaminated with coliform organisms and
containing 95 mg/L of nitrite (22 mg nitrate-nitrogen/L, equivalent to 3.5 mg
nitrate-nitrogen/kg/day). This infant recovered after a transfusion.
Simon et al. (1964) reported the results of a survey of infant
methemoglobinemia associated with nitrate-contaminated water occurring between
1956 to 1964 in the Federal Republic of Germany. A total of 745 cases were
located. For 306 of these cases, data were available on infant age and
frequency of diarrhea (an indication of bacteriological colonization). These
data indicated that 97.7% of the cases were 3-months-old or less and 53% had
diarrhea. Data were available on the nitrate content of the drinking water
for 249 of the 745 methemoglobinemia cases. In 83.8% of these cases, the
nitrate content of the drinking water was >100 mg/L (equivalent to 23 mg/L
nitrate-nitrogen); in 11.8% of the cases it was 50-100 mg/L (equivalent to
11-23 mg/L nitrate-nitrogen); and in 4.4% of the cases it was <50 mg/L
(equivalent to 11 mg/L nitrate-nitrogen). Only three cases of
methemoglobinemia (1.3%) were associated with water containing <20 mg/L
nitrate (equivalent to 4.5 mg/L nitrate-nitrogen). The authors indicated that
in each of these three cases, spinach and spinach juice were significant
sources of nitrate and nitrite, and in one case nitrite was known to be
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present in the water (concentration not reported). Therefore, the total dose
of nitrate plus nitrite in these cases was greater than predicted from just
the water intake.
Simon et al. (1964) measured methemoglobin levels in infants from three
towns in Germany. There were 89 infants receiving nitrate-free water,
38 infants receiving water containing 50-100 mg/L nitrate (11-23 mg nitrate-
nitrogen/L) and 25 infants receiving water containing greater than 100 mg/L
nitrate (>23 mg nitrate-nitrogen/L). A dose-response relationship between
nitrate concentration in the water and methemoglobin concentration was
apparent only in infants less than 3 months of age. In these infants, mean
methemoglobin level was 1% in the infants receiving nitrate-free water, 1.3%
in infants receiving 50-100 mg nitrate/L and 2.9% in the infants receiving
water containing nitrate in excess of 100 mg/L. Mean methemoglobin levels in
infants greater than 3 months of age were 0.8% (0-100 mg nitrate/L) or 0.7%
(>100 mg nitrate/L). No clinical signs of methemoglobinemia were detected.
Based upon their finding and reviews, the authors conclude that 100 mg/L
nitrate (23 mg nitrate-nitrogen/L) represents a safe level in water which is
free of bacteria or other contaminants.
Toussaint and Selenka (1970) supplied 34 healthy infants (under 3 months
of age) with formula prepared with water containing 150 mg N03/L (34 mg
nitrate-nitrogen/L) for 10 days. Average methemoglobinemia levels rose from
about 1% to about 2%-3% within 1-2 days, and then tended to stay steady for up
to 10 days. No clinical signs of methemoglobinemia were reported.
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Winton et al. (1971) studied a group of 111 infants who ingested varying
levels of nitrate. Sixty-three received a nitrate dose of less than
1 mg/kg/day (equivalent to 0.23 mg/kg/day nitrate-nitrogen), 23 were exposed
to 1-4.9 mg/kg/day (equivalent to 0.23-1.1 mg/kg/day nitrate-nitrogen), 20
were exposed to 5.0-9.9 mg/kg/day (equivalent to 1.1-2.2 mg/kg/day nitrate-
nitrogen) and 5 infants were exposed to 10-15.5 mg/kg/day (equivalent to
2.3-3.6 mg/kg/day nitrate -nitrogen). Only three infants appeared to have
methemoglobin levels above normal (0%-2.9%) and they were the youngest of the
five who had received more than 10 mg/kg/day. The highest methemoglobin level
(5.3%) was found in a 30-day-old baby who had received 15.5 mg/kg/day.
Assuming that a 4-kg infant consumes 0.64 liters of formula per day (Davidson
et al. 1975), these levels are equivalent to drinking water concentrations of
approximately 14-22 mg nitrate-nitrogen/L.
Shearer et al. (1972) examined the association between age and
methemoglobinemia in infants in south-central California. A total of 487
examinations were conducted on 256 infants over a period of 1 year.
Approximately 26% of infants aged 0-3 months had methemoglobin levels >3%,
while only 1% of infants older than 3 months had methemoglobin levels above
this level. These results indicate that young infants (up to 3 months of age)
were more likely to have elevated methemoglobin levels than older infants. No
clear association between nitrate intake in the diet and methemoglobin level
was detected.
Shuval and Gruener (1972) studied the association between drinking water
nitrate and methemoglobinemia in infants in Israel. The study population
consisted of 1,702 infants from an area in which the mean drinking water
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nitrate concentration was 70 mg/L (equivalent to 16 mg/L nitrate-nitrogen).
The control population consisted of 758 infants from an area with a water
supply containing 5 mg/L nitrate (equivalent to 1.1 mg/L nitrate-nitrogen).
There were essentially no differences in methemoglobin levels between the two
populations. The average level was 1.01% in the study area compared to 1.11%
in the control area. In both areas, average methemoglobin levels tended to
decrease as a function of age, from an average of about 1.33% in 0-2 month old
infants to 0.98% in infants 91 days or older. Young infants (1-90 days) with
diarrhea tended to have a somewhat higher average methemoglobin value (1.56%)
than average, but this difference was not apparent in older infants.
Jones et al. (1973) reported a case involving the situation surrounding a
previously healthy 10-week-old female baby who was admitted to the hospital
with "striking" cyanosis. Blood methemoglobin was 47.6%. Investigation
revealed that the child had been given one bottle of well water containing
110 mg/L of nitrate (25 mg nitrate-nitrogen/L) just prior to the acute
methemoglobinemia. The infant was subsequently fully recovered and was
discharged from the hospital within 1 week.
Gruener and Toeplitz (1975) fed formula containing varying levels of
nitrate to 104 infants for 5 days. On the first day of the study, all infants
were given formula made with water containing 15 mg/L nitrate (equivalent to
3.4 mg/L nitrate-nitrogen). For the next 3 days they were fed formula mixed
with water containing 108 mg/L nitrate (equivalent to 24 mg/L nitrate-
nitrogen). On the last day of the study the infants again received formula
prepared with water containing 15 mg/L nitrate. There was a slight but
significant rise in mean methemoglobin levels after the first day of exposure,
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although three individuals had especially high methemoglobin levels (6.9, 13.9
and 15.9%). There was a drop in mean methemoglobin levels to almost normal on
the 3rd day. These levels remained constant on the 4th day and decreased
further on the final day of the study, suggesting some adaptation.
Wurkert (1978) measured methemoglobin levels in 96 infants residing in
various locations across Germany (Rheinhessen) and also sampled and measured
nitrate levels in the drinking water. The majority (82%) of the infants were
under the age of 3 months. The methemoglobin concentrations in the study
population ranged from 0.33% to 14.15% with a mean of 2.30%. A significant
statistical correlation was reported between the concentration of nitrate in
the drinking water and methemoglobin concentration. Infants consuming
drinking water with a nitrate concentration between 0-11 og nitrate-nitrogen/L
(0-50 mg NOj/L) had methemoglobin levels ranging from 1.65% to 2.44%
(80 infants). Infants consuming water containing 12-23 mg nitrate-nitrogen/L
(51-100 mg N03/L) had methemoglobin values of 3.51 (12 infants) and those
consuming water containing nitrate at concentrations over 23 mg nitrate-
nitrogen/L (100 mg nitrate/L) had methemoglobin levels of 6.59% (4 infants).
Super et al. (1981) examined the association between drinking water
nitrate concentration and methemoglobin levels in 486 African infants. The
study area was divided into low and high nitrate regions according to whether
the drinking water nitrate-nitrogen concentration was <20 mg/L or >20 mg/L.
In infants living in the low nitrate region, 13% (38/293) had methemoglobin
levels of >3%. In infants living in the high nitrate region, 33% (64/193) had
methemoglobin levels of >3%. No further details on individual levels were
reported.
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Toussaint and Uurkert (1982) studied the methemoglobin levels of
96 infants in Germany as a function of the nitrate concentration in drinking
water. A clear dose - response relationship was detected, with mean
methemoglobin levels increasing from 1.7% in infants consuming 0-10 mg N03/L
(0-2.3 mg nitrate-nitrogen/L) to 6.6% in infants consuming >100 mg N03/L
(>23 mg nitrate-nitrogen/L). Levels tended to be higher in summer months than
in the winter, presumably because of greater water intake.
Hegesh and Shiloah (1982) studied the relationship of methemoglobinemia
to nitrate or nitrite ingestion in hospitalized infants with diarrhea. Blood
nitrate concentrations and methemoglobin levels were measured in 58 infants
with diarrhea and 130 controls without gastrointestinal disturbances.
Sixty-nine percent of the infants with diarrhea had methemoglobin levels above
those for the infants without diarrhea. The authors demonstrated that the
infants with diarrhea excreted up to 10 times more nitrate than they ingested,
while the controls excreted approximately 2.5 tines their nitrate intake. The
authors concluded that diarrhea alone can cause infant methemoglobinemia as
the result of endogenous nitrate formation.
Johnson et al. (1987) reported the case of a female infant, approximately
2 months old, who died following consumption of well-water containing 150 mg/L
nitrate-nitrogen (34.1 mg nitrate-nitrogen/L). The infant's blood
methemoglobin concentration was not reported, although she was described as
having previous episodes of cyanosis, vomiting and diarrhea. Prior to death
her blood was described as chocolate-brown in color.
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2. Methemoglobinemia in Older Children and Adults
Craun et al. (1981) conducted an epidemiologic study of 102 children aged
1-8 years in Washington County, Illinois. Sixty-four of the children were
from families consuming high-nitrate water <22-111 mg/L nitrate-nitrogen) and
38 children (controls) were from families consuming water containing less than
10 mg/L nitrate-nitrogen. In children aged 1-4 years, ingestion of increasing
doses of nitrate in water (from 25 to 500 mg NOj/24 hours) was found to result
in only a slight increase in methemoglobin levels (from 1.00% to 1.36%), a
difference which was not statistically significant. Essentially no dose-
dependent increase was detected in children aged 5-8 years, whose
methemoglobin values averaged around 0.90%-0.95% regardless of nitrate intake
from water.
In adults, methemoglobinemia has only been reported following accidental
ingestion of large doses of nitrite. Aquarmo et al. (1981) described a case
involving two laboratory technologists who salted their breakfast with a
laboratory salt shaker containing sodium nitrite. Symptoms appearing within
30 minutes included weakness, sweating, nausea, throbbing and a roaring sound
in the ears, palpitations, numbness and tingling. Both went to the emergency
room for treatment and were found to have cyanosis with methemoglobin levels
of 34% and 54%. Treatment with the antidote methylene blue lowered
methemoglobin levels to normal in 1 hour.
Valley and Flanagan (1987) reported several cases of methemoglobinemia in
humans who had consumed meat containing high levels of nitrite. In one event,
a 41-year-old woman and her 18-year-old son consumed meat containing
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15,000 ppm nitrite. Blood samples drawn at admission to the hospital revealed
23% methemoglobin in the woman and 7.7% in the son. Their recovery was
complete within 12 hours with no treatment other than oxygen. In another
case, a 36-year-old man became severely cyanotic and hypoxic 1 hour after
consuming meat containing 10,000 ppm nitrite. His blood methemoglobin
measured upon arrival at the hospital was 66%. Following treatment with
oxygen and methylene blue he recovered. Assuming consumption of about 0.2 kg
(0.4 lb) of contaminated meat, these cases probably involved intake of about
2,000-3,000 ng nitrite (about 8-12 mg nitrite-nitrogen/kg).
Kaplan et al. (1990) described 10 cases of severe methemoglobinemia in
adults who ingested a meal accidentally salted with NaN02. One person died
and two others were severely affected. Methemoglobin levels in these
individuals were 79% and 71%. All patients were tachypneic and cyanotic, and
some displayed hypotension, tachycardia, and altered levels of consciousness.
No estimate of dose was provided.
B. Teratogenicity and Reproductive Effects
Scragg et al. (1982) and Dorsch et al. (1984) conducted a case-control
study in the Mount Gambier region of South Australia to evaluate the
relationship between birth defects in children and maternal drinking water
parameters. They analyzed 218 case-control pairs identified from birth
records available from the years 1951 through 1979, matched by hospital,
maternal age, parity and month of delivery. Information about maternal water
source was available for each pair. A statistically significant increase in
the risk of birth defects (relative risk - 2.8) was reported for women
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consuming groundwater <5-15 mg/L nitrate) compared to women consuming
rainwater (less than 5 mg/L nitrate). The authors emphasized that their
results were limited by a number of factors, including possible sampling bias,
lack of nitrate exposure data and possible presence of other contaminants in
well-water (e.g., chemicals from local wood processing industries or
agricultural pesticides). On this basis the authors stated that "it would be
premature to interpret our case-control findings exclusively in terms of water
nitrate exposure."
Arbuckle et al. (1988) conducted a case-control study to determine the
relationship between nitrate concentration in drinking water and differing
rates of CNS birth defects in two areas of New Brunswick, Canada. One hundred
thirty cases of CNS defects identified from the high- and low-risk areas of
New Brunswick for the years 1973-1983 were included in the study. Each case
was matched with two randomly selected controls. Compared to a baseline
exposure level of 0.1 ppm nitrate in water, exposure to increasing levels of
nitrate in well water (1.25-26.0 ppm) was found to be associated with an
increased risk of birth defects (relative odds ratio - 2.3), but this was not
statistically significant. In contrast, exposure to increasing levels of
nitrate in public water (0.36-3.25 ppm) or spring water (0.23-16.7 ppm) was
found to result in decreased risk of birth defects (relative odds ratio - 0.39
and 0.0, respectively). These were also not statistically significant.
Aschengrau et al. (1989) performed a case-control study to investigate
the relationship between the levels of various chemicals in drinking water and
the incidence of spontaneous abortion in women. There were 286 cases and
1,391 controls selected from residents in or close to Boston. Exposure to
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nitrate in water (0.1-5.5 mg/L) was found to be associated with a decreased
risk of abortion (adjusted odds ratio — 0.5), and exposure to nitrite
(0.01-0.03 mg/L) was found to have no significant effect on risk (crude odds
ratio - 1.1-1.2).
C. Other Noncancer Effects
Inorganic nitrite ion is a direct-acting relaxant of smooth muscle
(Nickerson 1975, Moulds et al. 1981). The main effect is on vascular smooth-
muscle (especially the venous system) resulting in vasodilation and
hypotension. A single oral dose of 180 mg HaN02 given to an adult male
(corresponding to a dose of 0.5 mg nitrite-nitrogen/kg) produced no
significant effects as long as the individual was lying down. When raised to
a near-vertical position, a number of symptoms characteristic of marked
hypotension ensued, including sharply decreased venous pressure, decreased
systolic pressure, increased diastolic pressure, increased heart rate, and
deep respirations (Weiss et al. 1937). These symptoms disappeared when the
patient was returned to a reclining position. Doses of 30-60 mg of NaN02 are
typically employed for therapeutic purposes to treat angina pectoris
(Nickerson 1975) .
A variety of organic nitrates (e.g., acryl nitrate, nitroglycerin) also
produce these same effects, and are usually about 10- to 100-fold more potent
than the N02" ion. Inorganic nitrate ion does not produce the effect. The
mechanism of vasodilation appears to be by production of S-nitrosothiols which
stimulate guanylate cyclase activity in target cells (Herman et al. 1989),
perhaps through formation of nitric oxide (NO) (Ignarro 1990).
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d. Mut3ggiuc*,ty
No studies were located which investigated the in vivo incidence of
mutations in any human cell types as a function of nitrate or nitrite
exposure. However, it is known that ingestion of nitrate or nitrite can
increase the level of various N-nitroso compounds in the body, presumably
formed mainly as a result of reactions between nitrite and secondary amines in
the stomach (see below). For example, Stemmermann et al. (1981) detected
direct-acting mutagens in mucosa removed from the human stomach during
surgery. The mutagens were later identified as the N-nitroso derivatives of
three drugs (hydroxyzine hydrochloride, diazepam and cimetidine) that had been
administered to the patients.
E. Cancer
1. Epidemiological Studies
A number of epidemiological studies have been performed to determine if
ingestion of nitrate and/or nitrite is associated with increased risk of
cancer (especially stomach cancer) in humans. There are a number of factors
which tend to complicate the performance and interpretation of such studies:
• Nitrates and nitrites occur naturally in or are added to many foods,
so all humans are exposed through the diet. Intake levels may be
quite variable between individuals.
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•	The mechanism of carcinogenesis is presumed to involve irj vivo
formations of nitrosamines, so intake of nitrosable substrates
(mainly secondary amines) in the diet is also an important parameter
that may influence the observed risk levels.
•	Antioxidants such as vitamin C and vitamin E are thought to play a
protective role, so intake of these materials is also an important
variable that may decrease observed risk levels.
•	Treatment of food with nitrate or nitrite can sometimes lead to
formation of carcinogenic nitrosamines in the food itself before
ingestion. This can contribute cancer risk not properly
attributable to the nitrate or nitrite intake per se.
•	Exposure estimates are often indirect, and are usually available
only for the present time. However, cancer risk is more likely a
function of past exposure levels.
Some studies have attempted to deal with some of these factors, but many
studies have not. Such studies must be interpreted with due caution.
Geographical Correlation Studies
Most studies on cancer risk from nitrate/nitrite are geographical
correlation studies, in which average cancer incidence in an area is
correlated with average nitrate/nitrite levels in food or water in the same
area. This type of study is generally recognized as providing data that is
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useful in hypothesis formation, but not in establishing causal links. This is
because it is not possible to account for multiple risk factors, and no data
are collected to show that affected individuals actually had higher exposures
to nitrate or nitrite than the unaffected individuals. Several studies of
this type are summarized below.
Hill et al. (1973) reported that the death rate from gastrointestinal
cancer was higher than expected (SMR - 131 for males and 193 for females) in a
town in England where water contained 90 ppm nitrate (20 mg nitrate-
nitrogen/L). However, the death rates were not adjusted for the socioeconomic
status of the population. In a more careful study of the same population,
Davies (1980) adjusted stomach cancer death rates for difference in social
class distribution and occupation. After adjustment, the SMR was 97 for men
and 123 for women. These values were not significantly different from values
in a number of similar towns where nitrate levels in water were not elevated.
Colombia has one of the highest rates of mortality from stomach cancer,
especially among persons living at high altitudes in certain rural areas
(Haenszel and Correa 1975). Cuello et al. (1976) found that average nitrate
levels were higher in wells in the high risk areas (31 mg/L) than in the low
risk areas (5.6 mg/L), and that a higher percentage of people in the high risk
areas drank water from wells (56%) than in the low risk areas (24%). This
evidence of increased nitrate intake in the high risk areas was supported by
measurements of urinary nitrite excretion levels which were higher in the high
risk than the low risk areas.
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Zaldivar and Wetterstrand (1975) studied the correlation between death
from gastric cancer and the amount of nitrate fertilizers used in 25 provinces
of Chile. A highly significant positive correlation was found between cancer
incidence and fertilizer use both for the general population (P<0.00005) and
for farm workers (P<0.Q001). The authors interpreted these data to indicate
that use of nitrate fertilizers resulted in increased exposure to nitrate, and
that this lead to increased risk of gastric cancer by ia vivo formation of
nitrosamines. A similar association between stomach cancer mortality and use
of nitrate fertilizers in Chile was reported by Annijo and Coulson (1975).
Zaldivar and Wetterstrand (1978) studied the relation between death from
stomach cancer and nitrate levels in drinking water in urban areas from 25
provinces in Chile. Drinking water levels ranged from 0.0 to 30 ppm nitrate-
nitrogen/L. Weak positive correlations were found between nitrate levels and
cancer death rate for both men and women, although neither association was
statistically significant.
Juhasz et al. (1980) compared the stomach cancer incidence in 230
localities in the County of Szabolcs-Szatmar, Hungary, to the nitrate content
of the local drinking water. In general, localities with high nitrate levels
(defined as >100 ng N03/L) tended to have above average incidence of stomach
cancer, but many communities did not fit this pattern. It was concluded that
other factors caused stomach cancer in the low-nitrate areas.
Yang (1980) summarized epidemiological studies in several counties in
China where Incidence rates of esophageal cancer are very high (135/100,000).
Numerous factors were compared in high- and low-incidence areas in an effort
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Co identify risk factors. An association was found between increased
incidence and a number of dietary factors, including the use of water high in
nitrates and nitrites. In the high-risk areas, water was often from shallow
ponds that were heavily contaminated with micro-organisms and refuse.
Concentrations of nitrate and nitrite averaged 12.6 and 2.6 mg/L,
respectively, and the concentration tended to correlate with esophageal cancer
incidence rates. Other risk factors included ingestion of nitrosamines in
moldy food and pickled vegetables.
Gilli et al. (1984) performed a descriptive epidemiological study in
Italy in which the geographical pattern of gastric cancer was compared to
geographical variations in nitrate levels in drinking water. They found
positive association between nitrate levels and cancer risk, with a relative
risk of 13.7 for communities with water above 20 mg/L nitrate (4.5 mg nitrate-
nitrogen/L). This study did not control for other important risk factors for
gastric cancer (age, diet, alcohol consumption, occupation, socioeconomic
status, etc.), so the observed association may have been due to other factors.
Also there were large differences in community size, which makes local
mortality statistics extremely variable.
Beresford (1985) examined cancer mortality data in 253 urban areas in the
United Kingdom, adjusting death rates for variations in socioeconomic status
and population size. Drinking water nitrate levels were negatively associated
with stomach cancer mortality (that is, stomach cancer death rates were low
where nitrate levels were high).
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Forman et al. (1985) compared salivary levels of nitrate and nitrite in
over 800 healthy male and female volunteers (15-75 years old) from two regions
in the United Kingdom recognized as having distinctly different mortality
rates for gastric cancer. A written survey was used to evaluate socioeconomic
and dietary distinctions between individuals. Mean salivary nitrate and
nitrite concentrations in volunteers from 2 areas of the low risk region (208
and 157.3 nmol nitrate/mL; 124 nmol nitrite/mL and 95 nmol nitrite/mL) were
significantly greater (P<0.0Q01) than salivary levels of nitrate and nitrite
in 2 areas of the high risk region (107 nmol nitrate/mL; 69 and 51 nmol
nitrite/mL). Based on this, the estimated total daily intake of both nitrate
and nitrite was significantly greater in the low risk group (119.24 og
nitrate; 6.77 mg nitrite) than the high risk group (74.92 mg nitrate; 4.95 mg
nitrite).
Takacs (1987) investigated the relationships between incidence of
digestive system tumors and the nitrate content of drinking water in Hungary.
There was an apparent correlation between incidence and average nitrate
concentration across seven districts where water levels ranged from 90 to
230 mg N03/L, but a statistical test of trend or significance was not
reported.
The high incidence of esophageal cancer in Iran has been studied by a
Joint Iran-International Agency for Research on Cancer Study Group (1977).
Villages with different rates of esophageal cancer were surveyed for dietary,
work and personal habits. Their main foodstuffs were analyzed for volatile
nitrosamines, nitrate, nitrite and other compounds. Their drinking water was
tested for nitrate and nitrite. The average daily dietary intake of nitrate
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and nitrite was not significantly different for high- and low-incidence areas,
and nitrate and nitrite levels in water were not elevated in areas at high
risk for esophageal cancer.
Dutt et al. (1987) measured the nitrate content of a variety of
vegetables, fruits and meats consumed by the residents of Singapore. Based on
1982 statistics regarding food imports and production, average daily intake of
nitrate was calculated to be 215 mg/day. Two vegetables (Chinese cabbage and
kale) were particularly high in nitrate, contributing about 50% of the total.
The authors stated that the Malay and Indian subpopulations of Singapore
consumed less of these vegetables than the Chinese, and noted that the rate of
gastric cancer was lower for these two groups than for the Chinese. The
authors noted that this could be a chance association, since there are many
other factors that influence the incidence of gastric cancer.
Anquela et al. (1989) investigated the relation between the incidence of
gastric cancer and nitrite content of drinking water in nine areas in the
province of Soria, Spain. Water concentrations ranged from 0.4 to 11.8 mg
nitrite-nitrogen/L. There was a statistically significant (P<0.01) positive
association between nitrate level and mortality from gastric cancer. The
authors noted that highest rates occurred in grain-producing agricultural
regions, and that other factors related to agricultural work might also be
important.
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Cohort Studies
Fraser et al. (1980b, 1982) studied the association between inhalation
exposure to nitrate dust and the incidence of various cancers in fertilizer
workers in England and Wales. Men (age 35-64) identified from the 1961 and
1971 censuses as "manufacturers of fertilizers or chemicals for pest control"
were selected as the study populations. Jobs were coded according to a
protocol which estimated the frequency of exposure to nitrate-containing dust
as either frequent, infrequent, very infrequent or never. In the 1961 cohort,
the ratio of observed to expected deaths (0/E) from all causes was 0.82,
consistent with a "healthy worker" effect. Cancer deaths were also lower than
expected (0/E—0.85). Only intestinal cancer (0/E—1.33) and bladder cancer
(0/E-1.03) had ratios greater than 1.00, but these increases were not
statistically significant. In the 1971 cohort, the 0/E ratio for all cancers
was 1.32. No statistically significant increase in cancer mortality
statistics for any of the cancer types were detected, although the 0/E ratio
for esophageal (2.5), gastric (1.33), rectal (3.33) and bladder (1.41) cancers
were greater than 1.00 and were also greater than the ratios seen in the 1961
cohort. An apparent dose-response relationship (not statistically
significant) was noted in cancer deaths as a function of exposure duration
(0/E - 0.42 for "never" or "Infrequent", 1.31 for "infrequent" and 1.69 for
"frequent"). No statistically significant differences were observed between
groups exposed to different product categories. The authors concluded that
this study provided weak evidence of an association between frequency of
exposure to nitrate-containing dusts and increased risk of cancer mortality.
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Al-Dabbagh et al. (1986) studied mortality patterns in 1,327 male workers
in the production of nitrate-based fertilizers in England. Workers employed
in such capacity between 1946 and 1981 for at least 1 year were followed up
until 1981. Workers were classified into heavy, moderate or low exposure
groups based upon their estimated exposure to nitrate. Validation of exposure
was accomplished by measuring nitrate and nitrite concentration of saliva in
workers (30 per group). Nitrate concentrations in saliva were significantly
higher in workers with high (212.3 nraoles/mL) or moderate (206.8 nmoles/mL)
exposure to nitrate than in workers with no exposure to nitrates
(102.7 nmoles/mL). No significant increase in mortality was observed from
cancer or other diseases evaluated in either the entire cohort or the subgroup
of workers heavily exposed to nitrates.
Case Control Studies
Haenszel et al. (1973) reported an association between increased risk of
stomach cancer in Japanese immigrants to Hawaii and consumption of salt-
pickled vegetables (which contain nitrite) along with salted dried fish (which
contain nitrosable secondary amines) (Singer and Lijinsky 1976). This
suggests that one or both of these dietary components may be a risk factor for
stomach cancer, but the data are not adequate to draw firm conclusions.
Risch et al (1985) performed a case-control study on the relationship
between dietary history and the risk of stomach cancer in Canada. There were
246 cases and 246 controls, matched by age, sex and area of residence.
Dietary history was investigated by questionnaire, and intake of specific
dietary constituents (nitrate, nitrite, nitrosamine, vitamins, etc.) was
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calculated based on U.S. Department of Agriculture data on food composition.
Statistically significant associations were noted for intake of nitrite, along
with a number of other substances including smoked meats, unsaturated fats,
grains, chocolate, eggs, cream desserts and non-refrigerated foods. Intake of
fiber, citrus fruits and vitamin C resulted in a significant decrease in risk
of stomach cancer. Intake of nitrate also had an odds ratio significantly
less than 1.0. The authors concluded that nitrite intake is associated with
increased risk of stomach cancer.
Fontham et al. <1986) performed case-control studies to identify dietary
risk factors for chronic atrophic gastritis, which is a recognized precursor
to gastric cancer (Correa et al. 1976). A total of 93 hospital cases with
biopsy-confirmed chronic atrophic gastritis were compared to 186 age- and
sex-matched controls in the same hospital. Cases ingested less fruit,
vegetables and vitamin C, which was viewed as supporting intragastric
nitrosylation as the etiologic mechanism. Stomach pH was higher in cases than
controls (a condition favoring bacterial growth), but nitrate and nitrite
levels in gastric juice were not higher in cases than controls. This is in
contrast to an earlier study by Correa et al. (1979) on nitrite levels in
gastric juice of patients with atrophic gastritis from a region in Columbia
with a high gastric cancer incidence. In this population, many patients with
the disease had increased levels of nitrite in gastric fluid. This was
observed only in individuals with stomach pH above 5. For example, three
patients with stomach pH values of 7.1, 7.5 and 9.0 had stomach nitrite levels
of 193, 136 and 103 ppm, respectively. There was no clear relationship
between gastritis, stomach pH and stomach nitrate levels. The authors
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interpreted these data to indicate that bacterial growth in the stomach above
pH 5 could lead to the formation of nitrite, which would increase risk of
stomach cancer through formation of nitrosamines.
2. In Vivo Formation of Nitrosamines
Kamiyama et al. (1987) studied the concentrations of nitrate and
N-nitrosoaaino acids in the urine of inhabitants of two areas of Japan where
the risks of stomach cancer were low (33/100,000) and high (101/100,000)
respectively. Nitrate levels in urine were lower in the high risk area than
the low risk area (116 vs. 140 mg/person/day, P<0.07), but levels of
N-nitrosothiazolidine-4-carboxylic acid (NTCA) was significantly higher
(P<0.05). The authors interpreted these data to suggest that the residents of
the high-risk area had a greater potential for endogenous nitrosylation,
possibly due to the presence of a nitrosylation-inhibiting factor in the low
risk area.
Bellander et al. (1988) exposed four healthy adult volunteers to air
containing 0.3 mg/m3 of piperazine. When the volunteers ingested a diet low
in nitrate (40-70 mg/d), only one person had measurable levels of
nitrosopiperazine in the urine (0.36 Mg/32 hr). When ingesting a diet high in
nitrites (180-270 mg/day), nitrosopiperazine was detected in three of four
samples (mean - 1.2 jig/32hr). This was all found in the urine sample
collected 4 hours after the end of piperazine exposure.
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Moller et al. (1989) studied the influence of nitrate levels in water on
nitrosamine formation in a rural population in Denmark. Participants ingested
500 mg of proline in the evening, and a 12 hour urine sample was collected the
next morning and analyzed for N-nitrosoproline. In the whole cohort, there
was only a week positive association (P-0.08) between nitrate levels in water
and nitrosoproline excretion. This was probably due to the large variations
between people in the amount of nitrate intake from food. When the cohort was
separated into smokers and nonsmokers, nitrosolation was strongly associated
with nitrate intake in nonsmokers (P<0.01).
Zatonskl et al. (1989) studied the concentration of nitrate and
N-nitrosoamino acids in the urine of inhabitants of high- and low-risk areas
for stomach cancer in Poland. Urinary levels of nitrates were 1.4 higher in
the high-risk area (P<0.01). Levels of some nitrosoanino acids were not
different between the two groups, but levels of N-nitrososarcosine and
3(N-nitroso-N-methylamine)propionic acid were 3-4 fold higher in people from
the high risk area (P<0.02). When inhabitants were given doses of proline,
levels of nitrosoproline increased in the urine of high-risk but not low-risk
area inhabitants. The authors concluded that residents of the high-risk area
produced higher levels of endogenous nitrosamines than residents of the
low-risk area.
F. Summary
The principal adverse health effect associated with exposure of humans to
nitrate is methemoglobinemia in infants (age 0-3 months). This group is much
more susceptible than older children and adults because the pH of the infant
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stomach can often be sufficiently high (above pH 4) to permit growth of
bacteria that reduce nitrate to nitrite. Many studies have been performed on
the levels of nitrate in water that lead to clinically significant
methemoglobinemia in infants. These studies have shown that most cases are
associated with exposure to water containing 20 mg nitrate-nitrogen/L or
higher, and that no significant effects occur if water contains 10 mg nitrate-
nitrogen/L or less. In adults, methemoglobinemia has only been reported
following accidental ingestion of large quantities of sodium nitrite (usually
mistaken for table salt).
Only two studies are available on possible developmental or reproductive
effects of nitrate in humans. One study suggested that there might be an
increased risk of birth defects associated with ingestion of water containing
5-15 mg nitrate-nitrogen/L, but there were a number of other factors that
could also have been responsible for the observation. No significant relation
between nitrate levels in water and birth defects was detected in the second
s tudy.
Many epidemiological studies have been performed to determine if
ingestion of nitrate or nitrite are associated with increased cancer risk.
Some of these studies have noted increased cancer risk (especially
gastrointestinal cancer) in populations ingesting above average levels of
nitrate or nitrite in water or food. However, these studies are not adequate
to show that nitrate/nitrite caused the increased risk, since reliable dose-
response data are usually lacking, and there are many other risk factors for
gastrointestinal cancer that might have been responsible.
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VII. MECHANISMS OF TOXICITY
A.	Cardiovascular Effect
The acute pharmacological action of nitrite is to relax smooth muscles.
The relaxation is nonspecific and affects all smooth muscle irrespective of
its innervation or the nature of its responses to adrenergic, cholinergic, or
other types of agonists. However, nitrite does not prevent cells from
responding to an appropriate stimulus and its effect can be antagonized by any
drug that can activate the smooth muscle under consideration. Thus, nitrite
is a functional antagonist of norepinephrine, acetylcholine, histamine and
many other agents (Nlckerson 1975). The mechanism of this effect appears to
involve production of S-nitrosothiols which stimulate guanylate cyclase
activity in target cells (Herman et al. 1989), perhaps through formation of
nitric oxide (Ignarro 1990).
B.	Effect on Hemoglobin
Nitrite reacts with hemoglobin in the erythrocytes, oxidizing the iron of
hemoglobin to the ferric state. Ferric hemoglobin is called methemoglobin and
is unable to transport oxygen (Parks et al. 1981). During formation of
metHemoglobin, superoxide ion (02~) is also formed. The superoxide ion is
converted by superoxide dlsmutase to hydrogen peroxide (H202). This in turn
forms a catalase-H202 complex, for which additional nitrite may act as a
substrate and be oxidized to nitrate (Parks et al. 1981).
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C. Diuretic Effects
The diuretic effect of very high doses of nitrate in dogs was attributed
to enhancement of sodium excretion. This resulted in a net loss of sodium
from the body when nitrate was given as the sodium salt (Greene and Hiatt
1954). The rate of excretion of sodium administered with nitrate was much
greater than its rate of excretion when given with chloride. In the dog, as
much as 40% more sodium was excreted than was injected (as sodium nitrate),
bringing about a loss of extracellular fluid.
D. Effects Due to N-Nltrosation
N-Nitrosation of suitable nitrogenous substrates by nitrite in the
stomachs of laboratory rodents has been demonstrated by many investigators.
Substrates for N-nitrosation by nitrite include secondary amines (which form
nitrosamines), N-alkylureas, N-alkylcarbamates and simple N-alkylamides (which
form nitrosamides). Tertiary amines can also be nitrosated. Most of these
reaction products break down to release nitrosodinethylamine or
nitrosodiethylamine (Mirvish 1975).
The amount of nitroso compound produced in the stomach depends partly on
the nitrosation kinetics. For nitrosation to occur, nitrite is first
converted to HN02 (pK,-3.37). This explains why nitrosation is catalyzed by
acid. The reaction rate shows a maximum at pH 3.4. Nitrous acid is then
converted to an active nitrosating species, usually nitrous anhydride (N203)
(Mirvish 1975).
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Mirvish (1975) concluded che race of nitrosation is proportional Co the
concentration of amine and Che square of the concencration of nitrite.
Therefore, the race is affected more by an increase in nitrite than by an
increase in amine. The rate constants for many secondary amines have been
determined. The rate increases as the basicity of the amine decreases (as the
pKa of the amine decreases). Therefore, weakly basic amines react very
rapidly.
Thiocyanate increases the rate of nitrosation because the nitrosating
species nitrosothiocyanate (ON'NCS) is produced. The optimum pH for
nitrosation at high concentration of thiocyanate is 2.0. At this point,
nitrite becomes fully protonated. Below pH 2.0, Che rate falls because NCS"
becomes protonated. Thiocyanate-catalyzed nitrosation of amines will compete
favorably with the N203 mechanism shown above under three conditions: (1) at
pH less than 2.5, (2) ac high thiocyanate concentration, or (3) at low nitrite
concentration (Mirvish 1975).
Mirvish (1975) concluded that the rate of formation of N-nitroso
compounds in the stomach is proportional to the thiocyanate concentration as
well as the concentrations of amine and nitrite. Thiocyanate is secreted in
human saliva along with nitrate and swallowed into che stomach. Boyland and
Walker (1974) showed chac aC pH 1.5, 1.0 mM chiocyanace increases the rate of
che nitrosation reaction about 550 times. Human gastric juice is between pH 1
and 2.
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Ascorbic acid in the stomach reacts rapidly with nitrite to reduce it and
block the nitrosation reaction. Ascorbic acid is oxidized to dehydroascorbic
acid in the process. The blocking is most efficient under anaerobic
conditions (Archer et al. 1975).
E. Summary
Methemoglobinemia is the result of a nonenzymic reaction between
hemoglobin and nitrite, resulting in oxidation of Fe+2 to Fe+3. Nitrate does
not cause this reaction unless it is first reduced to nitrite.
Nitrite ion also acts directly on smooth muscle to cause relaxation. The
mechanism of this effect is not known precisely, but may involve activation of
guanylate cyclase.
Nitrite ion undergoes protonation in the stomach to form nitrous acid,
which may react with a variety of secondary and some tertiary amines to form
nitrosamines. This reaction is inhibited by ascorbic acid, and can be
increased by thiocyanate. Formation of nitrosamines is believed to be the
mechanism responsible for the hepatotoxic and carcinogenic effect observed in
animals exposed to high doses of nitrite and nitrosable substrates.
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VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS
The quantification of toxicological effects of a chemical consists of
separate assessments of noncarcinogenic and carcinogenic health effects.
Chemicals which do not produce carcinogenic effects are believed to have a
threshold dose below which no adverse, noncarcinogenic health effects occur,
while carcinogens are assumed to act without a threshold.
Quantification of Noncarcinogenic Effects
In the quantification of noncarcinogenic effects, a Reference Dose (RfD),
(formerly called the Acceptable Daily Intake (ADI)), is calculated. The RfD
is an estimate of a daily exposure to the human population that is likely to
be without appreciable risk of deleterious health effects, even if exposure
occurs over a lifetime. The RfD is derived from a No-Observed-Adverse-Effect
Level (NOAEL), or Lowest-Observed-Adverse-Effect Level (LOAEL), identified
from a subchronic or chronic study, and divided by an uncertainty factor(s).
The RfD is calculated as follows:
DfT) _ (NOAEt, PIT WWr)	 _ m_/k_ bw/day
R£D Uncertainty Factor(s) 	 mS/*S D /aay
Selection of the uncertainty factor to be employed in the calculation of
the RfD is based on professional judgment, while considering the entire data
base of toxicological effects for the chemical. In order to ensure that
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uncertainty factors are selected and applied in a consistent manner, the
Office of Drinking Water (ODW) employs a modification to the guidelines
proposed by the National Academy of Sciences (NAS 1977, 1980) as follows:
•	An uncertainty factor of 10 is generally used when good chronic, or
subchronic human exposure data identifying a NOAEL are available and
are supported by good chronic, or subchronic toxicity data in other
species.
•	An uncertainty factor of 100 is generally used when good chronic
toxicity data identifying a NOAEL are available for one or more
animal species (and human data are not available), or when good
chronic, or subchronic toxicity data identifying a LOAEL in humans
are available.
•	An uncertainty factor of 1,000 is generally used when limited or
incomplete chronic, or subchronic toxicity data are available, or
when good chronic, or subchronic toxicity data identify a LOAEL, but
not a NOAEL for one or more animal species are available.
The uncertainty factor used for a specific risk assessment is based
principally on scientific judgment rather than scientific fact and accounts
for possible intra- and interspecies differences. Additional considerations
not incorporated in the NAS/ODW guidelines for selection of an uncertainty
factor include the use of a less than lifetime study for deriving a RfD, the
significance of the adverse health effect and the counterbalancing of
beneficial effects.
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From the RfD, a Drinking Water Equivalent (DUEL) can be calculated. The
DWEL represents a medium specific (i.e., drinking water) lifetime exposure at
which adverse, noncarcinogenic health effects are not anticipated to occur.
The DWEL assumes 100% exposure from drinking water. The DWEL provides the
noncarcinogenic health effects basis for establishing a drinking water
standard. For ingestion data, the DWEL is derived as follows:
Body weight - assumed to be 70 kg for adult
Drinking water volume - assumed to be 2 liters per day for an adult
In addition to the RfD and the DWEL, Health Advisories (HAs) for
exposures of shorter duration (One-day, Ten-day and Longer-term) are
determined. The HA values are used as informal guidance to municipalities and
other organizations when emergency spills or contamination situations occur.
The HAs are calculated using a similar equation to the RfD and DWEL; however,
the KOAELs or LOAELs are identified from acute or subchronic studies. The HAs
are derived as follows:
DWEL
(RfD) x (Body Weight in kg)	
Drinking Water Volume in L/day
- 	 mg/L
where:
CNOAEL or LOAEL1 x (bw)
(UF) x (	 L/day)
- 	 ng/L
VIII-3

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Using che above equation, the following drinking water HAs are developed
for noncarcinogenic effects:
1.	One-day HA for	a 10-kg child ingesting 1 L water per day.
2.	Ten-day HA for	a 10-kg child ingesting 1 L water per day.
3.	Longer-term HA for a 10-kg child ingesting 1 L water per day.
A.	Longer-term HA for a 70-kg adult ingesting 2 L water per day.
The One-day HA calculated for a 10-kg child assumes a single acute
exposure to the chemical and is generally derived from a study of less than
7 days duration. The Ten-day HA assumes a limited exposure period of
1-2 weeks and is generally derived from a study of less than 30-days duration.
A Longer-term HA is derived for both the 10-kg child and a 70-kg adult and
assumes an exposure period of approximately 7 years (or 10% of an individual's
lifetime). A Longer-term HA is generally derived from a study of subchronic
duration (exposure for 10% of animal's lifetime).
Quantification of Carcinogenic Effects
The EPA categorizes the carcinogenic potential of a chemical, based on
the overall weight-of-evidence, according to the following scheme:
• Group A: Known Human f:arclnoften. Sufficient evidence exists from
epidemiology studies to support a causal association between
exposure to the chemical and human cancer.
V1II-4

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Group B: Probable Human Carcinogen. Sufficient evidence of
carcinogenicity in animals with limited (Group Bl) or inadequate
(Group B2) evidence in humans.
• Group C: Possible Human Carcinofen. Limited evidence of
carcinogenicity in animals in the absence of human data.
Group D: Not classified as to Human Carcinogenicity. Inadequate
human and animal evidence of carcinogenicity or for which no data
are available.
Group E: Evidence of Noncarcinogenicitv for Humans. No evidence of
carcinogenicity in at least two adequate animal tests in different
species or in both adequate epidemiologic and animal studies.
If toxicological evidence leads to the classification of the contaminant
as a known, probable or possible human carcinogen, mathematical models are
used to calculate the estimate excess cancer risk associated with the
ingestion of the contaminant in drinking water. The data used in these
estimates usually comes from lifetime exposure studies in animals. In order
to predict the risk for humans from animal data, animal doses must be
converted to equivalent human doses. This conversion includes correction for
noncontinuous exposure, less than lifetime studies and for differences in
size. The factor that compensates for the size difference is the cube root of
the ratio of the animal and human body weights. It is assumed that the
average adult human body weight is 70 kg and that the average water
consumption of an adult human is 2 liters of water per day.
VIII-5

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For contaminants with a carcinogenic potential, chemical levels are
correlated with a carcinogenic risk estimate by employing a cancer potency
(unit risk) value together with the assumption for lifetime exposure via
ingestion of water. The cancer unit risk is usually derived from a linearized
multistage model with a 95% upper confidence limit providing a low dose
estimate; that is, the true risk to humans, while not identifiable, is not
likely to exceed the upper limit estimate and, in fact, may be lower. Excess
cancer risk estimates may also be calculated using other models such as the
one-hit, Veibull, logit and probit. There is little basis in the current
understanding of the biological mechanisms involved in cancer to suggest that
any one of these models is able to predict risk more accurately than any
others. Because each model is based upon differing assumptions, the estimates
which were derived for each model can differ by several orders of magnitude.
The scientific data base used to calculate and support the setting of
cancer risk rate levels has an inherent uncertainty due to the systematic and
random errors in scientific measurement. In most cases, only studies using
experimental animals have been performed. Thus, there is uncertainty when the
data are extrapolated to humans. When developing cancer risk rate levels,
several other areas of uncertainty exist, such as the incomplete knowledge
concerning the health effects of contaminants in drinking water, the impact of
the experimental animal's age, sex and species, the nature of the target organ
system(s) examined and the actual rate of exposure of the internal targets in
experimental animals or humans. Dose response data usually are available only
for high levels of exposure, not for the lower levels of exposure closer to
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where a standard may be set. When there is exposure to more than one
contaminant, additional uncertainty results from a lack of information about
possible synergistic or antagonistic effects.
A. Noncancer Effects
1. Nitrate
Nitrate toxicity is due primarily to its conversion to nitrite, which
oxidizes the Fe+2 form of iron in hemoglobin to the Fe+3 state. This compound
(methemoglobin) does not bind oxygen, resulting in reduced oxygen transport
from lungs to tissues. Low levels of methemoglobin occur in normal
individuals, with typical values usually ranging from 0.5% to 2.0% (Winton
et al. 1971, NAS 1981). However, due to the large excess capacity of blood to
carry oxygen, levels of methemoglobin up to around 10% are not associated with
any significant clinical signs (Walton 1951, Winton et al. 1971, ECETOC 1988).
Concentrations above 10% may cause a bluish-color to skin and lips (cyanosis),
while values above 25% lead to weakness, rapid pulse and tachypnea (Jones
et al. 1973). Death may occur if methemoglobin values exceed 50%-60%.
Conversion of nitrate to nitrite is mostly mediated by bacteria in the
alimentary system. Consequently, the risk of methemoglobinemia from ingestion
of nitrate depends not only on the dose of nitrate, but also on the number and
type of enteric bacteria. In healthy adults, available data suggest that a
total of about 5% of a dose of nitrate is reduced to nitrite, mainly by
bacteria in the mouth (NAS 1981).
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Conversion of nitrate to nitrite may also occur in the stomach if the pH
of gastric fluid is sufficiently high (above pH 4) to permit bacterial growth.
This may occur in adults with diseases such as achlorhydria or atrophic
gastritis, and may also occur in some infants, since the infant gastrointes-
tinal system may have a pH high enough to permit growth of nitrate-reducing
bacteria. Reduction of nitrate in the stomach is believed to account for the
observation that infants (especially age 0-3 months), are most susceptible to
nitrate-induced methemoglobinemia. Risk may be particularly high in infants
who are exposed to water that is contaminated with bacteria, for two reasons.
First, ingestion of high levels of bacteria in water tends to promote
colonization of the gut with organisms that reduce nitrate to nitrite.
Second, bacteria in the gut can lead to gastroenteritis and diarrhea, which in
turn lead to increased nitrate intake through increased water consumption.
Most cases of methemoglobinemia in humans develop within several hours of
ingestion of a dose of nitrate (e.g., Comly 1945, Vigil et al. 1965, Miller
1971, Jones et al. 1973, Kaplan et al. 1990). Thus, methemoglobinemia is most
appropriately considered an acute effect. In the absence of repeated doses,
recovery is usually complete within several days (e.g., Donahoe 1949), and
children continuously exposed show little tendency for cumulative toxicity
(e.g., Toussaint and Selenka 1970). In fact, continuous exposure sometimes
appears to result in an adaptive response in which methemoglobin levels tend
to decrease (Gruener and Toeplitz 1975). On this basis, it is concluded that
a value protective for acute exposures will also be protective for longer-term
exposures. Therefore, only a single value for nitrate is required, and this
will serve as the DWEL as well as the One-day, Ten-Day and Longer-term HA
values. Derivation of this value is presented below.
VIII-8

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Table VIII-1 Summary of Studies on Methemoglobinemia In Infants



noael'
LOAEL

Reference
Route/Medium
Duration
mg/N/kg/d
mg N/L
mg N/kg/d
mg N/L
Cornblath and Hartman 1948
Oral/Water
2-18d
23
140
-
-
Bosch et al. 1950
Oral/Water
Continuous
1.6
10
3.4-8
21-50
Walton 1951
Oral/Water
Continuous
1.6
10
1.8-3.2
11-20
Simon et al. 1964
Oral/Water
Continuous
>3.7
>23
-
-
Toussalnt and Selenka 1970
Oral/Formula
10 days
5.4
34
-
-
Wlnton et al. 1971
Oral/Formula
Continuous
3.6
22
-
-
Shuval and Gruener 1972
Oral/Water
Continuous
2.6
16
-
-
Jones et al. 1973
Oral/Water
1 day
-
-
4.0
3.8
25
Gruener and Toeplitz 1975
Oral/Formula
5 days
-
-
24
Vurkert 1978
Oral/Water
Continuous
>3.7
>23

-
Toussalnt and Wurkert 1982
Oral/Water
Continuous
>3.7
>23
5 4
-
Johnson et al. 1987
Oral/Water
-
-
"

34
'Metherooglobin levels up to 10% are not considered adverse.

-------
RfD for Nitrate
Table VIII-1 lists studies which provide reliable quantitative data on
the NOAEL and LOAEL for methemoglobinemia in human infants. Concentrations of
methemoglobin less than 10% are not considered adverse, and doses that do not
produce levels over 10% are listed as NOAELs.
Based mainly on the studies by Bosch et al. (1950) and Walton (1951), the
NOAEL is taken to be 10 mg nitrate-nitrogen/L (1.6 mg nitrate-
nitrogen/kg/day). In most studies, the LOAEL is greater than 20 mg nitrate-
nitrogen/L (3.2 mg/kg/day), although a small number of cases have been noted
at exposure levels between 10 and 20 mg nitrate-nitrogen/L (Walton 1951).
Based on this NOAEL, the RfD for nitrate is calculated as follows:
RfD(N03) - 16 mE nitrate-nttrogen/kg/dav
- 1.6 mg nitrate-nitrogen/kg/day
where:
1.6 mg/kg/day- NOAEL (mg nitrate-nitrogen/kg/day) , based on
weight-of-evidence from studies in human infants
1 - Uncertainty factor. A factor of 10 is often
applied to a NOAEL in huaans to account for
possible variation in sensitivity among
individuals. In this case, the NOAEL is based
on studies in the most sensitive subpopulation,
so no uncertainty factor is required to adjust
for this.
VIII-10

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DMEL for Nitrate
Based on the RfD of 1.6 mg nitrate-nitrogen/kg/day, the DWEL for nitrate
is calculated as follows:
DWEL(NO ) - 1,6 nitrate'nitrofen/kf/dav
3	0.16 L/kg/day
- 10 mg nitrate-nitrogen/L
where:
1. 6 mg/kg/day - RfD
0.16 L/kg/day - Average fluid intake, based on consumption of
0.64 L/day by a 4-kg infant (Davidson et al. 1975)
Consideration for Other Possible Noncarcinogenic Effects of Nitrate
There are no convincing data in animals or humans that ingestion of
nitrate is associated with any adverse effect other than methemoglobinemia.
An epidemiological study in Australia (Dorsch et al. 1984) suggested that
there may be an increased risk of birth defects in women consuming groundwater
(which contains 5-15 mg/L of NOj) compared to rainwater (which contains
0-5 mg/L NOj), but this observation was not taken to constitute proof that
nitrate was responsible for the difference in risk. In a similar study in
Canada (Arbuckle et al. 1988), no significant association was detected between
nitrate intake and risk of birth defects. Also, studies in animals exposed to
high levels of nitrate do not indicate any increased incidence of reproductive
or teratogenic effects (FDA 1972a,b).
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2. Nitrite
Few data exist on nitrite toxicity in humans. Since nitrite is directly
toxic (i.e., does not require metabolic conversion by bacteria), toxic effect
levels are likely to be less dependent on age than for nitrate. Nevertheless,
there do appear to be important differences between infants, adults and
animals with respect to the ease of nitrite oxidation of hemoglobin and the
rate of methemoglobin reduction back to hemoglobin (Lukens 1987). On this
basis, it appears most appropriate to consider the infant the most sensitive
subpopulation for nitrite as well as nitrate toxicity. However, there are no
reliable quantitative data on the noncancer effects of nitrite ingestion by
humans, either for infants or adults. Thus, the RfD for nitrite must be
derived by (a) extrapolation of nitrite toxicity data in animals to nitrite
toxicity in humans, or (b) by extrapolation of nitrate toxicity data in humans
to nitrite toxicity in humans. These options are discussed below.
a. Extrapolation from Nitrite Data in Animals
Table VIII-2 summarizes studies in animals with reliable quantitative
data on the toxic effects of nitrite. As in humans (exposed to nitrate), the
principal adverse effect associated with exposure is methemoglobinemia. The
data are reasonably consistent across studies, and suggest that a dose of up
to about 10 mg nitrite-nitrogen/kg/day does not cause any adverse effects,
while doses of 20 mg/kg/day or higher can produce methemoglobinemia and other
related histological changes. However, a NOAEL of 10 mg nitrite-
nitrogen/kg/day is somewhat higher than doses of nitrate-nitrogen known to
produce methemoglobinemia in human infants (see Table VIII-1). Since the dose
VI11-12

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Table VIII-2 Summary of Studies on the
Noncancer Effects of Nitrite in Animals
Reference
Species
Route/
Medium
Duration
Endpoints
NOAEL*	LOAEL
mg N/kg/day rag N/kg/day
Shuval and Gruener 1977	Mice
Rats
Imaizumi et al. 1980	Rats
Druckrey et al. 1963	Rats
Shuval and Gruener 1972	Rats
Imaizumi et al. 1980	Rats
Chow et al. 1980	Rats
Rats
Rats
Til et al. 1988
Grant and Butler 1989
Shuval and Gruener 1972 Rats
Inai et al. 1979
FDA 1972c,d
Mice
Mice
Rats
Rabbits
Hamsters
Water
Water
Gavage
Water
Water
Water
Water
Water
Food
Water
Water
Gavage
3 weeks
6 weeks
1 dose
70 days
24 months
6 months
16 weeks
14 months
13 weeks
115 weeks
24 months
18 months
GD6-15
GD6-15
GD6-18
GD6-10
Met Hb
Met Hb
Met Hb
Met Hb
Met Hb/Growth/Mort.
Met Hb/Growth/Devel.
Met Hb/Histopathol.
Met Hb/Organ Weight
Met Hb/Histopathol.
Hematology
Histopathology (lung,
blood vessels)
Histopathology (secondary
to methemoglobinemia)
Repro/Teratogen.
2.6
54
5
20
20
50
4
17
4.6
2
4.6
5.3
40
81
10
40
40
50
20
20
41

-------
Table VIII-2 (Continued)
Reference
Species
Route/
Medium
Duration
Endpoints
NOAEL*
mg N/kg/day
LOAEL
rag N/kg/day
Sleight and Atallah 1968
Guinea pigs
Water
100-240d
Repro/Teratogen.
180
192
Druckrey et al. 1963
Rats
Water
3 gen
Repro/Teratogen.
20
-
Shuval and Gruener
1972a,b
Rats
Water
Gest/Lact
Repro/Body Weight
-
54
Globus and Samuel 1978
Mice
Gavage
GD0-18
Repro/Develop.
3.4
-
Hugot et al. 1980
Rat
Food
3 gen
Growth
-
90
Roth et al. 1987
Rats
Water
Gest/Lact
Growth, Neonatal post
(lactation)
Fetal Hb
10
44
20
Shlobara 1987
Mice
Gavage
GD6-15
Repro.
8
16
Roth and Smith 1988
Rats
Water
Gest/Lact
Pup growth, Pup hlsto.
-
40
Shlmada 1989
Mice
Water
GD7-18
Repro/Growth/
Teratogenicity
49

*The NOAEL for methemoglobinemia Is taken to be 10%.

-------
of nitrite to the infant cannot exceed the dose of nitrate (and is presumably
somewhat less), these data indicate that animals are not as sensitive to
nitrite as human infants. Therefore, extrapolation of nitrite toxicity data
from animals to humans does not appear to be prudent.
b. Extrapolation from Nitrate Toxicity Data in Humans
The dose of nitrite formed in the body from an ingested dose of nitrate
can be calculated as follows:
Dose of Nitrite - Dose of Nitrate x Percent Conversion
Using this approach, the NOAEL for nitrite in humans may be estimated from the
NOAEL for nitrate, if percent conversion is known.
The percent conversion for older children and adults is estimated to be
about 5% (NAS 1981), but a reliable NOAEL for methemoglobinemia in this group
is not available. Even if the NOAEL for nitrate were known, an estimate of
the NOAEL for nitrite in this group might not be protective for infants, due
to the more rapid oxidation and slower reduction of hemoglobin in infants
(Lukens 1987).
Ideally, the NOAEL for nitrite should be based on the NOAEL for nitrate
in infants times the percent conversion in infants. While the NOAEL is known
with reasonable accuracy (i.e., 10 mg nitrate-nitrogen/L), there is no
reliable information on the percent of nitrate conversion to nitrite in
infants.
VIII-15

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In che absence of data, the NOAEL for nitrite may be estimated by
assuming a conversion rate in infants, It is important to note that, in this
case, it is not conservative to assume 100% conversion of nitrate to nitrite;
rather, the smaller the assumed conversion, the lower the estimated dose of
nitrite. For example, based on a NOAEL of 10 mg/L for nitrate-nitrogen, the
NOAEL for nitrite would be 10 mg/L if 100% conversion is assumed, but only
1 mg/L if 10% conversion is assumed. Based on the data which indicate that
conversion of nitrate to nitrite is at least 5% in adults (NAS 1981), and
knowing that nitrate reduction in infants is significantly greater than in
adults due to the presence of gastrointestinal bacteria, the conversion rate
is estimated to be at least 10% in infants. This is likely to be
substantially lower than the actual conversion rate in individuals who are
susceptible to methemoglobinemia, since they generally have a high
gastrointestinal content of bacteria that reduce nitrate to nitrite.
Employing this percent conversion, the RfD and DWEL for nitrite may be
calculated as follows:
RfD for Nitrite
RfD(N02) - 1.6 mg N03-nitrogen/kg/day x 0.1 me NO^-nitrogen
mg N03-nitrogen
- 0.16 mg nitrite-nitrogen/kg/day
where:
1.6 mg/kg/day - RfD for nitrate in human infants
0.1 - Conservative estimate of fraction of nitrate reduced to
nitrite kg infants.
VIII-16

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DUEL for Nitrite
DWELfNO \ M 0.16 nitrlte-nltro^en/k^/day
2	0.16 L/kg/day
- 1.0 mg nitrite-nitrogen/L
where:
0.16 mg/kg/day - RfD for nitrite
0.16	L/kg/day - Average fluid intake, based on 0.64 L/day by a
4-kg infant (Davidson et al. 1975)
Consideration of Other Possible Noncancer Effects of Nitrite
Studies in animals provide little evidence of noncancer effects other
than methemoglobinemia from ingestion of nitrite. No evidence of reproductive
or teratogenic effects has been noted in several studies, except at doses that
produce severe iron deficiency in the dam's milk (Roth et al. 1987). A few
reports identify histological changes in the lung (Shuval and Gruener 1972),
coronary arteries (Shuval and Gruener 1977), spleen (Fritsch et al. 1980) and
adrenal cortex (Til et al. 1988), but none of these effects appear to result
in any functional impairments at doses that produce methemoglobinemia.
B. Cancer
1.	Weight-of-Evidence Evaluation
Most studies of nitrate or nitrite ingestion by humans or animals have
yielded negative or equivocal evidence of carcinogenicity. A number of
VIII-17

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studies have shown chat animals fed nitrite along with very high doses of
nitrosable substrates have increased incidence of tumors. The USEPA is
currently evaluating these data, but a weight-of-evidence classification has
not yet been assigned for either nitrate or nitrite.
2. Quantification of Cancer Risk
The USEPA has not performed quantitative cancer risk calculations for
either nitrate or nitrite.
C.	Existing Guidelines and Standards
The World Health Organization recommends a limit of 10 mg/L nitrate -
nitrogen and less than 1 mg/L nitrite-nitrogen in drinking water (WHO 1984).
D.	Susceptible Subpopulations
Infants are at greatest risk of developing methemoglobinemia from nitrate
exposure. This increased susceptibility is related to at lease four factors
(Phillips 1971). First, fetal hemoglobin is more readily oxidized than
hemoglobin in adults. At birth, between 60% and 80% of the hemoglobin remains
in the immature form of the molecule, hemoglobin F. This decreases to
approximately 30% by the third month of life (Pisciotta et al. 1959). Second,
the infant has a transitory physiologic deficiency of methemoglobin reductase
or its cofactor, NADH (reduced nicotinamide adenine dinucleotide), which are
necessary to maintain iron in its reduced state (Ross and Desforges 1959).
Third, on a weight basis, infants consume over five times as much water (the
VIII-18

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most important source of nitrate in the etiology of methemoglobinemia) as
adults (Burden 1961). Finally, relative achlorhydria in the very young favors
overgrowth of nitrate-reducing bacteria in the stomach (Walton 1951).
Individuals with altered physiological states or with either hereditary
or acquired disease may also be predisposed to the development of nitrite- or
nitrate-induced methemoglobinemia. These include adults with reduced gastric
acidity (including those being treated for peptic ulcer or individuals with
chronic gastritis or pernicious anemia) and individuals with a rare,
hereditary lack of NADH or methemoglobin reductase activity in their red blood
cells (Scott 1960). This hereditary enzyme deficiency seems also to account
for the somewhat increased incidence of methemoglobinemia among Alaskan
Eskimos and Indians (Scott and Hoskins 1958).
Pregnant women may also be more susceptible than average adults to
nitrate-induced methemoglobinemia. Skrivan (1971) reported that methemoglobin
levels tended to increase during pregnancy, reaching a maximum of about 9%-10%
between 30-35 weeks of gestation. The basis for the increase was not
identified, but might be due to a reduced rate of methemoglobin reduction. No
evidence of cyanosis was observed in the pregnant women, and no significant
differences in methemoglobin levels were detected between women with normal
pregnancies and those with premature deliveries or abortions. Although these
observations do not indicate that the elevation of methemoglobin during
pregnancy is a risk factor for abnormal births, pregnant women may be more
likely to experience increased methemoglobin levels following nitrate/nitrite
exposure.
VIII-19

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E. Summary
Epidemiological studies and case reports from the United States and
elsewhere indicate that clinically significant methemoglobinemia does not
occur in infants who ingest water containing 10 mg nitrate/nitrogen/L or less.
This concentration corresponds to an average daily intake of 1.6 mg nitrate -
nitrogen/kg/day by an infant, and this is taken to be the Reference Dose (RfD)
for nitrate for an infant. This value is also protective for older children
and adults, since infants are the most sensitive subpopulation.
Data are not available to identify the NOAEL for nitrite in infants.
Based on the assumption that infants convert at least 10% of ingested nitrate
to nitrite, the NOAEL is estimated to be at least 1 mg nitrite-nitrogen/L.
Based on this, the RfD for nitrite is 0.16 mg nitrite-nitrogen/kg/day for an
infant. This value is also protective for older children and adults, since
they are somewhat less susceptible to nitrite-induced methemoglobinemia than
infants.
The USEPA is currently evaluating the evidence that ingestion of nitrate
or nitrite may be associated with increased risk of cancer. Cancer
weight-of-evidence categories have not yet been assigned.
VIII-20

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IX. REFERENCES
Al-Dabbagh, S.D. Forman and D. Bryson. 1986. Mortality of nitrate fertilizer
workers. Bri. J. Ind. Med. 43: 507-515.
Alavantic D, Sunjevark I, Pecevsk J, Bozin D, and G Cerovic. 1988a. In vivo
genotoxicity of nitrates and nitrites in germ cells of male mice. I.,
Evidence for gonadal exposure and lack of heritable effects. Mutation
Research, 203:689-695.
Alavantic D, Sunjevark I, Cerovic G, Bozin D, and Pecevski J. 1988b. In vivo
genotoxicity of nitrates and nitrites in germ cells of male mice. II.
Unscheduled DNA synthesis and sperm abnormality after treatment of spermatids.
Mutation Research 204:697-701.
Anderson LM, Giner-Sorolla A, Greenbaum JH, et al. 1979. Induction of
reproduction system tumors in mice by N6-(methylnitros)-adenosine and a
tumorigenic effect of its combined precursors. Int. J. Cancer.
24(3):319-322.
Anquela JM, et al. 1989. Correlacion del riesgo de cancer gastrico en la
provincia de soria con el contenido de nitratos en las aquas de bebida. Rev.
Esp. Enf. Ap. Digest 75(6):561-565.
Aquanno JJ, Chan K-M, Dietzler DN. 1981. Accidental poisoning of two
laboratory technologists with sodium nitrite. Clinical Chemistry
27:1145-1146.
Arbuckle TE, Sherman GJ, Corey PN, Walter D, and Lu B. 1988. Water nitrates
and CNS birth defects: A population-based case-control study. Arch. Environ.
Health. 43(2)-.162-167.
Archer MC, Tannenbaum SR, Fan T-Y, Weisman M. 1975. Reaction of nitrite with
ascorbate and its relation to nitrosamine formation. J. Natl. Cancer Inst.
54:1203-1205.
Armijo R, Coulson AH. 1975. Epidemiology of stomach cancer in Chile. X.
The role of nitrogen fertilizer. Int. J. Epidemiol. 4:301-309.
Asahina S, Friedman MA, Arnold E et al. 1971. Acute synergistic toxicity and
hepatic necrosis following oral administration of sodium nitrite and secondary
amines to mice. Cancer Res. 31:1201-1205.
Aschengrau A, Zierler S, Cohen A. 1989. Quality of community drinking water
and the occurrence of spontaneous abortion. Archives of Environmental Health
44:283-290.
Astill BD, Mulligan LT. 1977. Phenolic antioxidants and the inhibition of
hepatotoxicity from N-dimethylnitrosamine formed in situ in the rat stomach.
Food Cosmet. Tox. 15:167-171.
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Bartholomew BA, Hill MJ, Hudson MJ, Ruddell WSJ, Walters CL. 1980. Gastric
bacteria, nitrate, nitrite and nitrosamines in patients with pernicious anemia
and in patients treated with cimetidine. In: Walker EA, Castegnaro M,
Griciute L, Porzsonyi M, eds. N-nitroso compounds: analysis, formation and
occurrence, IARC Scientific Publication No. 31. Lyon, France: International
Agency for Research on Cancer pp. 595-608.
Bellander T, Osterdahl BG, Hagmar L. 1988. Excretion of
N-Mononitrosopiperazine after low level exposure to piperazine in air:
effects of dietary nitrate and ascorbate. Tox. App. Pharmacol. 93:281-287.
(In Spanish; Summary in English)
Beresford SAA. 1985. Is nitrate in the drinking water associated with the
risk of cancer in the urban U.K.? International Journal of Epidemiology.
14(1):57 - 63.
Bergman F, Wahlln T. 1981. Tumour induction in Syrian hamsters fed a
combination of amino pyrine and nitrit. Acta. Path. Microbiol. Scand. Sect. A
89:241-245.
Bloomfield RA, Hersey JR, Welsch CW, Garner GB, Muhrer ME. 1962. Gastric
concentration of nitrate in rats. J. Anim. Sci. 21:1019.
Bosch HM, Rosenfield AB, Houston R, Shipman HR, Woodward FL. 1950. J. Amer.
Water Works Assoc. 42:161. Cited in Simon C, Manzke H, Kay H, Mrowetz G.
1964. Uber Vorkommen, Pathogenese und Moglichkeiten zur Prophylaxe der durch
Nitrit verursachten Methajnoblobinamie. Z. Kinderheilkd. 91:124-138. (In
German; summary in English)
Boyland E, Walker SA. 1974. Effect of thiocyanate on nitrosation of amines.
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APPENDIX 1
EPA RESPONSES TO REVIEW COMMENTS
PROVIDED BY THE SCIENCE ADVISORY BOARD
The Drinking Wacer Subcommittee (now a full committee) of the USEPA Science
Advisory Board (SAB) performed a thorough review of the Final Draft Criteria
Document for Nitrate/Nitrite (dated May, 1987). Review of comments from the
SAB were forwarded to the administrator (Hon. Lee M. Thomas) by Richard
Griesemer and Norton Nelson, chairmen of the SAB, in a letter dated May 11,
1987. This appendix summarizes the comments by the SAB, and provides EPA's
responses.
Comment:
The documentation and its presentation are incomplete and confusing, thus
creating difficulties in reaching conclusions on matters of scientific
interpretation. For example, the observations of Walton are used as a basis
for determining acceptable levels for a ten-day health advisory. The
Subcommittee is particularly concerned about the weight given the Walton
study. The document describes the study in the summary with little or no
interpretation of the underlying study design, but its importance merits an
expert review by an epidemiologist. The study needs to be fully described in
the body of the document along with conclusions as to its limitations and
relative importance. Further, the same critical approach should be followed
for all studies that are crucial to the development of proposed standards.
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Response:
The organization of Che text has been revised to facilitate the Cask of data
interpretation. The description of the Walton study has been expanded to
provide greater detail on the nature of the study and its limitations.
Similar detail has been added to other key studies. The Walton study is
considered to be only one of many studies in humans which support che proposed
standard for nitrate, and the text has been revised to make this clear.
Comment:
Public health standards setting involves the examination and selection of
appropriate margins of safety. The Safe Drinking Water Act and its later
Amendments specify that EPA address this issue. It is especially critical
here since the document cites the National Academy of Sciences report
(Drinking Water and Health, 1977) that remarks on the narrow margin of safety
for nitrates. In the Criteria Document for Nitrate and Nitrite, the Agency
selects a margin of safety that excludes, for all practical purposes,
protection of sensitive members of the population, namely, infants with
gastrointestinal disease. The staff should clarify it technical rationale for
developing a margin of safety, as well as the need to include or exclude
particular subgroups of the population, for example, those individuals who are
genetically disposed to only slowly reduce the methemoglobin.
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Response:
EPA believes chat the standard of 10 mg/L contains an adequate margin of
safety to protect sensitive members of the population, namely, infants with
gastrointestinal disease. This conclusion is supported by numerous analyses
of the scientific literature. For example, both the World Health Organization
(Guidelines for Drinking Water and Other Supporting Information, 1984, WHO,
Geneva, pg 128-134) and the Canadian Department of Health and Welfare
(Guidelines for Canadian Drinking Water Quality, Supporting Documentation,
1978, pg. 419-431, Minister of Supplies and Services, Canada, 1980) concluded
that infant methemoglobinemia has not been reported where drinking water
contains less than 10 mg/L nitrate.
Comment:
The Office of Drinking Water should set a single public health standard for
the contribution from both nitrate and nitrite.
Response:
EPA agrees and proposes a combined nitrate/nitrite MCLG of 10 mg/L.
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The subcommittee notes that the document limits the consideration of exposure
to oral ingestion of drinking water. The document should attempt to place
into perspective the contributions of drinking water to total human health
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exposure by age group (particularly the endogenous sources); ac present, there
is little opportunity to determine to what extent Agency actions for drinking
water will provide overall public health protection. The Subcommittee
recommends that the Office of Drinking Water also expand the chapter on
alternate pathways and sources of exposure to provide a more comprehensive
analysis of relative exposure, and that the section on the quantification of
toxicological effects examine in detail the contribution of nitrate and
nitrite in drinking water to the risk of disease in various populations.
Response:
EPA has analyzed nitrate/nitrite exposure through various pathways. That
analysis is described in a document entitled "Estimated National Occurrence
and Exposure to Nitrate/Nitrite in Public Drinking Water Supplies". Available
information indicates that adults who consume nitrate at the proposed standard
of 10 mg/L would receive approximately 50% of their nitrate from drinking
water and 50% from the diet.
Comment;
A data gap exists for reproductive and developmental effects that the document
should cite. If reproductive and developmental toxicity data are not
available from either reliable human epidemiologic studies, from concretely
relatable human experience, or from valid state-of-the-art animal studies,
this fact itself must be stated. Furthermore, when developmental toxicity
data are available from animal studies, then both the NOEL and the magnitude
of the most likely margin of safety between that animal NOEL and probable
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human exposure needs to be stated. A second point is the relationship of the
vulnerability of the conceptus and the mother to the agent. If the agent in
question produces developmental toxicity only at, or very near to, maternally
toxic dose levels in an animal study, this needs to be stated as does the more
hazardous situation wherein an agent produces adverse effects on the conceptus
in the absence of any maternal homeostatic effect.
Response:
The revised criteria document contains summaries of numerous reproductive
studies of nitrate and nitrite in animals which indicate there is low risk of
teratological or developmental effects except at exposure levels that produce
marked maternal methemoglobinemia. The EPA believes these data are adequate
to show that reproductive effects are not the most sensitive endpoint of
nitrate/nitrite toxicity. The few studies that have been reported in humans
are too limited to draw firm conclusions. The text has been revised to expand
on this important topic.
Comment:
There is a carcinogenic potential of these inorganic ions in chemical
combination with naturally occurring substances. The Subcommittee recommends
that the Office of Drinking Water present a conclusion on the current
knowledge about the potential for impacts of nitrosated materials from nitrate
and nitrite in drinking water. The Criteria Document may require further
revision if new information arises about this subject. Specifically, it is
desirable to consider the case of splenic sarcomas in male rats which appear
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Co be related to compounds that produce methemoglobinemia. The Subcommittee
also recommends that the nitrosamine issue (endogenous formation) could
warrant a separate EPA position paper as some nitrosamines are likely to be
human carcinogens.
Response:
The Criteria Document has been revised to provide detailed review of studies
on the carcinogenicity of nitrate alone, nitrite alone, and nitrite plus
nitrosable substrates, as well as the potential to form nitrosamines in vivo.
The relationship between splenic tumors and methemoglobinemia has been
investigated, and there does not appear to be a strong link. Bus and Popp
(1987) noted that the only compounds which have been found to produce splenic
toxicity and carcinogenicity are structurally related to aniline, suggesting
that the key chemical requirement is an aromatic amine. The mechanism of
toxicity/carcinogenicity by such compounds is not known, but is suspected to
involve oxidation of phenylhydroxylamine (a metabolite of aniline produced in
the liver) to nitrosobenzene in the erythrocyte, a reaction that results in
methemoglobin formation. However, it is the ability to form nitrosobenzene,
not methemoglobin, that appears to be responsible for damage to the
erythrocyte and subsequent toxicity/carcinogenicity to the spleen. It should
be noted, that except for methemoglobin, nitrate/nitrite exposure usually
produces only mild effects on erythrocytes, and there is no evidence that
nitrate/nitrite result in nitrosobenzene formation or splenic toxicity.
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