The Risk Assessment Information System

Toxicity Profiles

Formal Toxicity Summary for NITRATES

NOTE: Although the toxicity values presented in these toxicity profiles were correct at the time they were produced, these values are subject to change. Users should always refer to the Toxicity Value Database for the current toxicity values.

EXECUTIVE SUMMARY
1. INTRODUCTION
2. METABOLISM AND DISPOSITION
2.1 ABSORPTION 2.2 DISTRIBUTION 2.3 METABOLISM 2.4 EXCRETION
3. NONCARCINOGENIC HEALTH EFFECTS
3.1 ORAL EXPOSURES 3.2 INHALATION EXPOSURES 3.3 OTHER ROUTES OF EXPOSURE 3.4 TARGET ORGANS/CRITICAL EFFECTS
4. CARCINOGENICITY
4.1 ORAL EXPOSURES 4.2 INHALATION EXPOSURES 4.3 OTHER ROUTES OF EXPOSURE 4.4 EPA WEIGHT-OF-EVIDENCE 4.5 CARCINOGENICITY SLOPE FACTORS
5. REFERENCES

AUGUST 1995

Prepared By: Andrew Francis, M.S., DABT, Chemical Hazard Evaluation Group, Biomedical Environmental Information Analysis Section, Health Sciences Research Division, *, Oak Ridge, Tennessee.

Prepared for: Oak Ridge Reservation Environmental Restoration Program.

*Managed by Martin Marietta Energy Systems, Inc., for the U.S. Department of Energy under Contract No. DE-AC05-84OR21400.

EXECUTIVE SUMMARY

Nitrates are produced by natural biological and physical oxidations and therefore are ubiquitous in the environment (Ridder and Oehme 1974). Most of the excess nitrates in the environment originate from inorganic chemicals manufactured for agriculture. Organic molecules containing nitrate groups are manufactured primarily for explosives or for their pharmacological effects (Stokinger 1982). Exposure to inorganic nitrates is primarily through food and drinking water, whereas exposure to organic nitrates can occur orally, dermally, or by respiration (Stokinger 1978). The primary toxic effects of the inorganic nitrate ion (NO3-) result from its reduction to nitrite (NO2-) by microorganisms in the upper gastrointestinal tract (Johnson and Kross 1990, Bouchard et al. 1992). Nitrite ions can also be produced with organic nitrate exposure; however, the primary effect of organic nitrate intake is thought to be dependent on the production of an active nitric oxide (NO-) radical (Waldman and Murad 1987). Organic nitrates are metabolized in the liver resulting in an increase in blood nitrites (Murad 1990). Nitrates and nitrites are excreted primarily in the urine as nitrates (Hartman 1982).

The primary toxic effect of inorganic nitrates is the oxidation of the iron in hemoglobin by excess nitrites forming methemoglobin. Infants less than 6 months old comprise the most sensitive population (Hartman 1982, Bouchard et al. 1992). Epidemiological studies have shown that baby formula made with drinking water containing nitrate nitrogen levels over 10 mg/L can result in methemoglobinemia, especially in infants less than 2 months of age. No cases of methemoglobinemia were reported with drinking water nitrate nitrogen levels of 10 mg/L or less (Bosch et al. 1950, Walton 1951, Shuval and Gruener 1972). A secondary target for inorganic nitrate toxicity is the cardiovascular system. Nitrate intake can also result in a vasodilatory effect, which can complicate the anoxia resulting from methemoglobinemia (Ridder and Oehme 1974). Decreased motor activity was reported in mice given up to 2000 mg nitrite/L in drinking water, and persistent changes in EEG recordings were observed in rats exposed to 100 to 2000 mg nitrite/L in drinking water. However, exposure of rats to 3000 mg nitrite/L in drinking water for 2 years did not result in any gross or microscopic changes in brain tissue. The data indicate that these central nervous system effects are not related to methemoglobin levels (Shuval and Gruener 1972).

The importance of the primary and secondary targets are reversed with organic nitrates, several of which have long been used for their vasodilatory effects in the treatment of angina pectoris in humans (Murad 1990). Large doses of organic nitrates, however, can also produce methemoglobinemia (Andersen and Mehl 1973). Epidemiological studies have shown that chronic or subchronic exposure to organic nitrates results in the development of tolerance to the cardiovascular effects of these compounds. This apparent biocompensation has caused serious cardiac problems in munitions workers exposed to organic nitrates when they are suddenly removed from the source of exposure (Carmichael and Lieben 1963).

An epidemiological study correlated the number of congenital malformations of the central nervous system and musculoskeletal system of babies with the amount of inorganic nitrate in the mother's drinking water (Dorsch et al. 1984). Other studies, however, do not support these associations, and the presence of unidentified teratogenic factors in the environment could not be ruled out. Inorganic nitrate and nitrite have been tested for teratogenicity in rats, guinea pigs, mice, hamsters, and rabbits. No teratogenic responses were reported; however, fetotoxicity attributed to maternal methemoglobinemia was observed at high doses (4000 mg nitrate/L in drinking water) (Sleight and Atallah 1968, Shuval and Gruener 1972, FDA 1972a, b, c).

A Reference Dose (RfD) of 1.60 mg/kg/day (nitrate nitrogen) for chronic oral exposure was calculated from a NOAEL of 10 mg/L and a LOAEL of 11-20 mg/L in drinking water, based on clinical signs of methemoglobinemia in 0-3-month-old infants (Bosch et al. 1950, Walton 1951). It is important to note, however, that the effect was documented in the most sensitive human population so no uncertainty or modifying factors were used (EPA 1994).

The possible carcinogenicity of nitrate depends on the conversion of nitrate to nitrite and the reaction of nitrite with secondary amines, amides, and carbamates to form N-nitroso compounds that are carcinogenic (Bouchard et al. 1992). Experiments with rats have shown that when given both components, nitrite and heptamethyleneimine, in drinking water, an increase in the incidence of tumors occurs (Taylor and Lijinsky 1975). Human epidemiological studies, however, have yielded conflicting evidence. Positive correlations between the concentration of nitrate in drinking water and the incidence of stomach cancer were reported in Columbia and Denmark (Cuello et al. 1976, Fraser et al. 1980). However, studies in the United Kingdom and other countries have failed to show any correlation between nitrate levels and cancer incidence (Forman 1985, Al-Dabbagh et al. 1986, Croll and Hayes 1988). Nitrate has not been classified as to its carcinogenicity by the EPA, although it is under review (EPA 1994).

1. INTRODUCTION

Nitrate (NO3-) (CAS No. 014797-55-8) is an inorganic anion resulting from the oxidation of elemental nitrogen. It is an essential nutrient for plant protein synthesis and plays a critical role in the nitrogen cycle of soil and water. Nitrates are produced by natural biological and physical oxidations and therefore are ubiquitous in the environment (Ridder and Oehme 1974). Most nitrate compounds are strong oxidizing agents and some can react violently with oxidizable substances and may explode if exposed to heat or shock (Sax and Lewis 1989).

Organic molecules containing nitrate groups are manufactured primarily for explosives or for their pharmacological effects (Stokinger 1982). Most of the excess nitrates in the environment originate from inorganic chemicals manufactured for agriculture. Farmers often apply fertilizer in the form of ammonium or sodium nitrate in excess to their crops. When the concentration of nitrates in the soil is higher than the plants can use, the excess nitrates appear in the surface and ground waters and are often found in drinking water, especially in rural agricultural areas served by wells.

Ammonia from animal waste and septic tanks can be oxidized to nitrate by soil bacteria under aerobic conditions. This can also be a significant source of nitrate in surface and groundwater especially near areas of concentrated animal populations, such as feedlots and dairy barns (Bouchard et al. 1992). The groundwater contamination also depends on the type and thickness of the soil, the amount of precipitation, irrigation, vertical flow, dissolved oxygen concentration, and electron donor availability. The groundwater in the agricultural southeastern United States is not very vulnerable to NO3 contamination, whereas it is a serious problem in parts of the midwest (Spalding and Exner 1993). In addition to drinking water, dietary sources of nitrates include compounds used in meat curing processes and nitrates in vegetables. High concentrations of nitrates in vegetables can reflect the overapplication of nitrate-containing fertilizers (Ridder and Oehme 1974, Phillips 1971).

Inorganic nitrate can be reduced to nitrite (NO2-) by the microflora in saliva and the gastrointestinal tract. Nitrite is thought to be responsible for most of the toxic effects observed with excess nitrate ingestion (Johnson and Kross 1990, Bouchard et al. 1992).

2. METABOLISM AND DISPOSITION

2.1. ABSORPTION

Inorganic nitrates are primarily absorbed through the gastrointestinal system as a mixture of nitrates and nitrites (Bouchard et al. 1992). Some organic nitrates can also be absorbed unchanged through the skin, gastrointestinal tract, mucous membranes, and lungs (Stokinger 1982). Nitrates and nitrogen oxides, which can be oxidized to nitrates, occur as organic products of photochemical smog and as inorganic aerosols in the atmosphere (NAS 1981). These substances can be absorbed through the respiratory system. The daily nitrate dose/person via respiration in the Los Angeles area has been estimated at about 500 µg nitrate-nitrogen (Fan et al. 1987).

2.2. DISTRIBUTION

Nitrates and nitrites are absorbed by the various routes into the general blood circulation and are transported to all parts of the body. Radioactive tracer experiments have shown that nitrates are distributed evenly among body organs, and the rate of distribution is dependent on blood flow (Parks et al. 1981). Animal experiments have shown that nitrites can cross the placental barrier and affect the fetus (Shuval and Gruener 1972).

2.3. METABOLISM

Nitrates are reduced to nitrites by the microflora in saliva and the gastrointestinal system (Hartman 1982, Ridder and Oehme 1974, Bouchard et al. 1992). The in vivo reduction of nitrates to nitrites depends on conditions that are subject to wide variations including the number and type of microflora present in the saliva and the gastrointestinal tract and the pH of the stomach. Gastric pH is higher in infants less than 6 months old and during some gastrointestinal infections (gastroenteritis), thereby favoring the reduction of nitrates (Bouchard et al. 1992). Nitrites absorbed into the blood are rapidly oxidized to nitrates. Nitrites have been shown to be oxidized to nitrates at the rate of more than 50% in 10 minutes at a concentration of 2 to 3 nanomoles/L of blood in mice and rabbits. A catalase-hydrogen peroxide system has been proposed as the oxidation mechanism (Parks et al. 1981).

Organic nitrates, which are absorbed intact and are used to relieve angina attacks, undergo reductive hydrolysis by the action of hepatic glutathione-organic nitrate reductase, forming a more water soluble organic molecule and inorganic nitrites. The kinetics of this reduction is dependent upon the organic nitrate molecule, the route of entry, and the hepatic blood flow. Most pharmacological doses of organic nitrate can undergo denitration during one circulation through the liver. Oral doses, some of which are absorbed into the portal circulation, are formulated to saturate the hepatic enzymes to facilitate a more prolonged prophylaxis against angina attacks (Murad 1990).

2.4. EXCRETION

Nitrates and nitrites are excreted in the urine primarily as inorganic nitrates. Small quantities of nitrates are excreted in the saliva, where they are subject to reduction to nitrites by microorganisms in the salivary ducts resulting in the recycling of a mixture of nitrates and nitrites in the gastrointestinal system (Hartman 1982).

3. NONCARCINOGENIC HEALTH EFFECTS

3.1. ORAL EXPOSURES

3.1.1. Acute Toxicity

3.1.1.1. Human

Nitrites formed from nitrates by the microflora in the salivary ducts and gastrointestinal system are primarily responsible for the toxic effects observed after nitrate ingestion (Fan et al. 1987, Bouchard et al. 1992). Inorganic nitrates, if not reduced to nitrites, are not toxic at concentrations found in drinking water, vegetables, and cured meats. Their physicochemical effects have been compared to the effects of sodium chloride in humans (Fan et al. 1987).

An excess of nitrites produced by the reduction of organic or inorganic nitrates can oxidize the iron in hemoglobin from ferrous to ferric, forming methemoglobin (Craun et al. 1981, Hartman 1982, Bouchard et al. 1992). This is the primary toxic effect of inorganic nitrate ingestion, and infants less than 6 months old comprise the most sensitive population. This sensitivity is due to the presence of more easily reduced fetal hemoglobin, a higher population of reducing bacteria in the stomach due to a higher gastric pH, lower enzymatic capacity to reduce methemoglobin, and a predisposition to gastrointestinal infections that tend to favor populations of reducing bacteria (Bouchard et al. 1992). Nitrites also have a vasodilatory effect that can further complicate the problem of methemoglobinemia-induced anoxia (Ridder and Oehme 1974).

Bosch et al. (1950) correlated the incidence of infant methemoglobinemia with the nitrate concentration of drinking water from Minnesota wells. The water was found to contain from 10 to greater than 100 mg nitrate-nitrogen/L. No cases of methemoglobinemia were found with baby formula made with well water containing 10 mg or less nitrate-nitrogen/L. The infants were less than 2 months of age in 90% of the methemoglobinemia cases. An epidemiological study by Walton (1951) analyzed all recorded cases of infant methemoglobinemia in 37 states. The occurrence of the condition was found to be primarily due to the ingestion of baby formula prepared with nitrate contaminated water. A total of 214 cases could be compared to nitrate concentrations in drinking water. No cases were recorded with drinking water containing 10 mg nitrate-nitrogen/L or less. Five cases were reported in infants exposed to 11-20 mg/L, 36 cases in those exposed to 21-50 mg/L, and 173 cases in infants exposed to greater than 50 mg/L nitrate-nitrogen. Additional studies have supported these observations.

Methemoglobin levels in 1702 infants with water supplies averaging 15.8 mg nitrate-nitrogen/L (70 mg nitrate/L) were compared with 758 infants with water supplies averaging 1.2 mg nitrate-nitrogen/L (5 mg nitrate/L) (Shuval and Gruener 1972). No cases of methemoglobinemia were reported, and only slight differences in methemoglobin levels were observed. No changes were observed in infants more than 90 days old. Infants with diarrhea had slightly increased methemoglobin levels (1.78%) compared to normal healthy infants (1.16%), and infants on a diet high in vitamin C-rich foods were observed to have slightly lower levels (1.19% compared to 1.30% methemoglobin). Only 6% of the children in this study were fed powdered formula made with the tap water. Knotek and Schmidt (1964) reported subclinical methemoglobinemia in infants fed on formula made with nitrate-rich tap water. Nitrate-induced infant methemoglobinemia persists today, especially in rural farming areas where reliance on well water is prevalent. Johnson et al. (1987) reported a fatal case of methemoglobinemia resulting from the feeding of powdered infant formula prepared with well water that was found to contain about 150 ppm nitrate nitrogen to an 8-week-old infant.

Organic nitrates are well known for their vasodilatory effects and have been used for the treatment of angina pectoris. Although nitrite release from organic nitrates accounts for the formation of methemoglobin, the vasodilatation effect of organic nitrates does not depend on the liberation of the nitrite groups (Stokinger 1982). The production of an active nitric oxide radical is thought to lead to the dephosphorylation of the light chain of myosin and the relaxation of smooth muscle (Fung et al. 1992, Murad 1990, Waldman and Murad 1987). Headache, dizziness, and weakness may also be experienced and is associated with the cardiovascular effects. Organic nitrates can also produce a drug-induced rash in susceptible people. The usual oral dose of most organic nitrates for the relief of angina symptoms is about 10-40 mg, 2 to 4 times daily. Specific organic nitrates that are given orally for their vasodilation effects include nitroglycerin, isosorbide dinitrate, erythrityl tetranitrate, and pentaerythritol tetranitrate. Peak effects usually occur in 60-90 minutes after oral administration and last 3-6 hours (Murad 1990).

3.1.1.2. Animal

Methemoglobinemia, which can lead to anoxia and death in extreme cases, is the primary acute toxic effect of oral exposure to inorganic nitrates in all animals tested. Ruminant animals are most susceptible. This effect is extremely variable since it depends on a number of factors including the conversion of nitrates to nitrites; the ability of the various animals to enzymatically reduce methemoglobin; the amount of vitamins A, C, D, and E in the diet; and the nutritional state of the animal. Acute nitrate toxicity in cattle has been reported following the ingestion of water containing 500 ppm or more nitrate or feed containing 5000 ppm or more nitrate. Methemoglobinemia is caused by the conversion of the nitrates to nitrites; however, high levels of nitrates have also been reported to result in gastroenteritis, diarrhea, diuresis, and petechial hemorrhages on the pericardium (Ridder and Oehme 1974). Dogs have sustained a plasma level of 24 mEq nitrate/L (336 mg nitrate-nitrogen/L) following gavage with sodium nitrate in water with no evidence of methemoglobinemia. Slight increases in glomerular filtration rates and renal plasma flow were observed, and hyperexcretion of chloride leading to hypochloremia, alkalosis, and digestive disturbances were reported. Dehydration occurred in some dogs as a result of the gastrointestinal problems and a diuretic effect of nitrates (Greene and Hiatt 1954). Dogs have also been given 20,000 ppm nitrate in their diet without any apparent adverse effects (Ridder and Oehme 1974). Rats have shown no effects after a dietary nitrate concentration of 10,000 ppm (PHS 1962). Pigs are even more resistant to nitrate poisoning but have developed methemoglobinemia after ingesting food or drinking water containing nitrites converted from nitrates by microflora in the food or water before ingestion (toxic dose listed as 88 mg nitrite/kg body weight) (Ridder and Oehme 1974). Potassium nitrate oral LD50 values of 3750 mg/kg for rats and 1901 mg/kg for rabbits and sodium nitrate oral LD50 values of 2680 mg/kg for rabbits have been reported (Sax and Lewis 1989).

Organic nitrates can also produce methemoglobinemia in animals, which contributes to the overall toxic response resulting in reduced average time to death. Oral LD50 values have been reported in rats for propylene glycol 1,2-dinitrate (PGDN) (250 mg/kg) and triethylene glycol dinitrate (TEGDN) (1000 mg/kg). PGDN also causes ataxia, lethargy, and respiratory depression in rats. TEGDN can also result in rats being hyperactive to auditory and tactile stimulation. Moderate increases in alkaline phosphatase and creatine kinase activities were reported following PGDN treatment (Stokinger 1982, Andersen and Mehl 1973).

3.1.2. Subchronic Toxicity

3.1.2.1. Human

Elevated methemoglobin as a result of subchronic exposure to high dietary or drinking water nitrate levels has been reported in school age children. Methemoglobin levels 2-5 times the levels seen in children with drinking water nitrate levels <10 mg nitrate-nitrogen/L were reported in groups of school children (total number 517) in the Soviet Union consuming water with 180 and 204 mg nitrate-nitrogen/L (Diskalenko 1968). In another study of 21 children 12-14 years old, there was a 7-fold increase in methemoglobin levels observed between the children exposed to a drinking water nitrate-nitrogen concentration of 23 mg/L compared to 2 mg/L (Subbotin 1961, Craun et al. 1981). Wide individual variations in responses to high dietary or drinking water nitrate levels are reported. Methemoglobin formation depends on the microflora conversion of nitrates to nitrites, the age and nutritional state of the individual, and the amount of vitamin C in the diet (Craun et al., 1981).

No increase in methemoglobin was observed in a group of 64 Illinois children consuming drinking water containing 22-111 mg nitrate-nitrogen/L compared with 38 children consuming water with <10 mg nitrate-nitrogen/L. The children were from 1-8 years of age. Evidence was presented in this study that indicated the length of exposure was less important than the concentration of nitrate in the water during the previous 24 hours before sampling blood for methemoglobin and also less important than the age of the children. The methemoglobin levels in the highest dose groups (201-500 mg nitrate estimated intake/ previous 24 hours) were significantly higher in the 1-4 age group than in the 5-8 age group. These differences, however, were not considered biologically significant (Craun et al. 1981). Toxic health hazards in humans, except for methemoglobinemia, as a result of subchronic high inorganic nitrate exposure are undocumented (Moller et al. 1989).

The subchronic administration of organic nitrates results in the development of tolerance to the cardiovascular effects of these compounds. This effect is independent of entry route and creates limitations in the treatment of angina symptoms and potentially serious problems for workers in munitions and dynamite industries (see Sect. 3.2.2.1.) (Stokinger 1982, Elkayam et al. 1992, Colucci et al. 1981).

3.1.2.2. Animal

One problem with inorganic nitrate studies in animals is the different rates of conversion of nitrate to nitrite seen in animals when compared to humans. Rats have a much lower nitrate to nitrite conversion rate than humans, which complicates interpretation and extrapolation of results. For this reason, Til et al. (1988) examined the toxicity of nitrite in a 90-day study in rats. Groups of 10 male and 10 female 6-week-old Wistar rats were given 100, 300, 1000, and 3000 mg potassium nitrite/L in drinking water. Potassium levels were equated in all groups by the addition of potassium chloride. Both tap water and tap water plus potassium chloride control groups were used. The estimated average intake of potassium nitrite was reported as 0, 8.9, 24.6, 77.5, 199.2 and 0, 10.9, 31.1, 114.4, 241.7 mg/kg/day for males and females, respectively. All animals appeared healthy during the entire 13-week study. Decreased food consumption and weight gain was seen in males at the high dose, and decreased drinking water consumption was reported in both sexes at the 3000 mg/L dose and at the 1000 mg/L dose in males. Methemoglobin was significantly (P <0.01) increased in both sexes at the high dose. Slight changes in erythrocyte parameters were also noticed, including decreased hemoglobin concentration and packed-cell volume and erythrocyte counts in the 1000 and 3000 mg/L groups. The high dose also resulted in an increase in plasma urea levels in males and a slight decrease in plasma alkaline phosphatase activity in both sexes although significantly only in females. The relative weights of the kidneys were increased in both sexes at the 3000 mg/L dose; however, histopathological examinations were negative. Thorough autopsies on all animals were performed, and the histopathological examinations revealed a dose-related hypertrophy of the adrenal zona glomerulosa in both sexes. The hypertrophy was correlated with previously reported changes in urinary steroid excretion in rabbits and humans following nitrite ingestion and with the vasodilating properties of nitrite.

A sedative effect was reported in groups of 57 black 6J male mice given drinking water containing 1500 and 2000 mg nitrite/L. A significant decrease in motor activity was measured in a special activity box designed for this purpose. Lower doses, 100 mg/L, and 1000 mg/L did not show the sedative effect. The decreased activity remained after methemoglobin levels were reduced to near normal following vitamin C administration, thereby indicating that the sedative effect may be independent of the methemoglobinemia. A subchronic study measuring brain electrical activity was designed to study this effect. Recordings were made using implanted electrodes to measure possible central nervous system effects in groups of 3-month-old male rats receiving 0, 100, 300, and 2000 mg/L sodium nitrite in drinking water. Recordings were made before the treatment began, during the 2 months of treatment, and for 4 months following cessation of the treatments. Alterations in the EEG recordings were observed in all treated groups. The changes persisted in the three highest groups during the observation period. Some recovery was noted in the low dose group; however, diffused spikes and sharp waves remained for the entire period. It was concluded that subchronic sodium nitrite ingestion in drinking water may result in persistent brain electrical changes in rats (Shuval and Gruener 1972).

3.1.3. Chronic Toxicity

3.1.3.1. Human

Toxic health hazards in humans, except for methemoglobinemia, as a result of chronic high inorganic nitrate exposure are undocumented (Moller et al. 1989).

The chronic administration of organic nitrates results in the development of tolerance to the cardiovascular effects of these compounds. This effect is independent of entry route and creates limitations in the treatment of angina symptoms and potentially serious problems for workers in munitions and dynamite industries (see Sect. 3.2.2.1.) (Stokinger 1982, Elkayam et al. 1992, Colucci et al. 1981).

3.1.3.2. Animal

Groups of 8 male rats were given 0, 100, 1000, 2000, or 3000 mg sodium nitrite/L in drinking water for 2 years. No significant differences in mortality, growth, or development were reported. A dose-related increase in methemoglobin levels was observed, but no significant differences were noticed in hemoglobin levels. Histological examination revealed no pathological changes in pancreas, adrenal, or brain tissue. However, pathological changes were reported with increased frequency at the higher doses in the lungs and heart. The observed changes included dilated bronchi, fibrosis and emphysema in the lungs, and fibrosis and degenerative foci in the heart. The coronary arteries in the high dose group were reported to be much thinner and dilated than expected in animals of their age. The high dose group was estimated to have received 250-350 mg sodium nitrite/kg body weight/day (60 mg nitrite-nitrogen/kg/day) (Shuval and Gruener 1972).

Druckrey et al. (1963) gave rats 100 mg sodium nitrite/kg/day (20 mg nitrite-nitrogen/kg/day) in a lifetime drinking water study. Elevated methemoglobin was reported in treated animals, but no other treatment-related hematologic or histologic effects were observed.

3.1.4. Developmental and Reproductive Toxicity

3.1.4.1. Human

An epidemiological study by Dorsch et al. (1984) involved 218 babies born in rural south Australia with congenital malformations. The babies were matched individually as to hospital, maternal age, parity, and date of birth with an equal number of normal babies. The nitrate concentrations in the water sources used in the homes during pregnancy were determined or estimated. Significantly (>95% confidence level) increased relative risk for malformations of the central nervous system and musculoskeletal system in babies was associated with mothers that used drinking water containing 5 - >15 ppm nitrate. Individuals using rainwater (<5 ppm nitrates) for drinking water were given a relative risk of 1.0; those exposed to water containing 5-15 ppm were found to have a relative risk of 2.8; and the individuals exposed to water containing >15 ppm nitrates had a relative risk of 4. Neural tube defects had the strongest association (relative risk of 3.5). Unidentified teratogenic factors that might be present in the water, diet, or environment could not be eliminated as causative or contributing factors.

3.1.4.2. Animal

Groups of 12 pregnant rats were given 2000 or 3000 mg/L sodium nitrite in drinking water. A control group of seven pregnant rats were given tap water. The dams in the 2000 mg/L group developed methemoglobinemia and decreased hemoglobin compared to controls and nonpregnant rats. No deformities were reported in any of the groups, and the birth weight of the pups was comparable to the controls. The pups of the treated groups had decreased growth rates, the fur was thin and lacked luster, and survival was decreased (mortality was 6, 30, and 53% for controls, 2000, and 3000 mg/L, respectively). The pups did not show abnormally high methemoglobin in either of the treated groups, although hemoglobin levels were about 20% less than the control group. Fetal blood nitrite levels following doses of 2.5 to 50 mg/kg sodium nitrate given orally to the dams were measured in a subsequent experiment. Elevated fetal blood nitrite and methemoglobin levels were reported after a lag of about 20 minutes. The threshold of transplacental transfer of nitrite was reported to be at a sodium nitrite dose of 2.5 mg/kg (Shuval and Gruener 1972).

Groups of 3 to 6 female guinea pigs were given 0, 300, 2500, 10,000, or 30,000 ppm potassium nitrate in drinking water for 143 to 204 days. The daily intake of nitrate nitrogen was calculated to be 12, 102, 507, and 1130 mg/kg body weight for the 300, 2500, 10,000, and 30,000 ppm doses in drinking water, respectively. Five animals or less were kept in one cage including one male rabbit per cage. The daily food and water consumption were measured and the animals were weighed each week. The number of litters produced, live births, and fetal deaths during the treatment period were reported. A decrease in the number of litters (2 treated, 8 control) and the number of live births (2 treated, 31 control) were reported for animals in the 30,000 ppm dose group, which were treated for 204 days. One animal in this group died with four mummified fetuses in utero. The fetal deaths were attributed to hypoxia due to maternal methemoglobinemia. Food and water consumption were comparable in all groups and weight gains were normal. No significant gross or microscopic lesions were reported in the reproductive organs. In a parallel experiment, 3-6 female guinea pigs per group were given 300, 1000, 2000, 3000, 4000, 5000, or 10,000 ppm potassium nitrite in drinking water corresponding to 18, 45, 154, 182, 192, 244, and 577 mg nitrite nitrogen/kg body weight/day, respectively. Treatment duration varied from 100 days for 4000 ppm to 240 days for 300 ppm. In this case, the number of litters produced per female (0.7 treated, 2 control) and live births per female (1.7 treated, 7.8 control) were decreased at 4000 ppm, and 4 fetal deaths were reported versus 1 in the control group. No live births at 5000 or 10,000 ppm were recorded.

Inflammatory cervical and uterine lesions and degenerative placental lesions were reported in females with dead fetuses. The relative percent reproductive performance was calculated taking into consideration the number of females, the average number of days under treatment, and the total number of live births. The control group was assumed to be 100%. This indicator dropped from 80% at 3000 ppm to 41% at 4000 ppm and to 0.0% at 5000 ppm. Food and water consumption was near normal at all doses. Decreased weight gain was seen with the highest dose of potassium nitrite. Methemoglobin levels were about 20% of the available hemoglobin in the 10,000 ppm group (Sleight and Atallah 1968). Male fertility was apparently not greatly affected since conception occurred at all doses. Wide variations were reported in the results of these two experiments, such as no live births at 5000 or 10,000 ppm in one study, but 2 live births at a dose of 30,000 ppm in the other study.

Sodium and potassium nitrate and nitrite were tested in mice, rats, hamsters, and rabbits for teratogenicity in studies sponsored by the Food and Drug Administration. The oral doses for sodium nitrate given through gestation were up to 400 mg/kg for groups of 20 to 26 mice and hamsters and up to 250 mg/kg for groups of 20 to 26 rats and 10 to 13 rabbits. Potassium nitrate up to 400 mg/kg for mice, 1980 mg/kg for rats, 280 mg/kg for hamsters and 206 mg/kg for rabbits was given. No effects were reported in any treated group on nidation, maternal or fetal survival, or incidence of soft or skeletal tissue abnormalities. Sodium and potassium nitrite at doses up to 23, 10, 23, and 23 mg/kg were given throughout gestation to mice, rats, hamsters, and rabbits, respectively. Although there was no teratogenic response in any group, an indication of slightly delayed skeletal maturation, especially in the ribs and skull, was observed in rats at the highest dose (10 mg/kg) (FDA 1972a, b, c).

Groups of 22 to 28 female rats were given 0.0125, 0.025, or 0.05% sodium nitrite in their diet from 14 days before breeding through gestation and lactation. Offspring were given sodium nitrite at the same dietary level as their parents for up to 90 days of age. Males were also given the same doses 14 days before breeding. The negative control group contained 35 females. No significant decreases in body weight or food consumption were reported from birth through lactation in any of the treated groups. No malformations or significant effects were noted on reproductive performance at any of the doses tested. Offspring mortality, however, was increased at the middle and high dose up to day 24 after birth, after which no further increase in deaths occurred. The period of increased mortality was concurrent with a transient period of decreased weight gain and delayed swimming development. There were no effects reported on post-weaning development, weight gain, food consumption, mortality, 90-day brain and eye weights, and tests of adult behavior (Vorhees et al.1984).

3.1.5. Reference Dose

3.1.5.1. Subchronic

A subchronic RfD for nitrates is not available.

3.1.5.2. Chronic

ORAL RfDc: 1.60 mg/kg/day (EPA 1994)

UNCERTAINTY FACTOR: 1

MODIFYING FACTOR: 1

NOAEL: 10 mg nitrate-nitrogen/L of drinking water

LOAEL: 11-20 mg nitrate-nitrogen/L of drinking water

CONFIDENCE:

Study: High

Data Base: High

RfD: High

VERIFICATION DATE: 08/22/90

PRINCIPAL STUDY: Walton, G. (1951); Bosch, H.M. et al. (1950)

COMMENTS: The NOAEL was obtained from the amount of nitrate-nitrogen in well water used to prepare formula for infants. It is based on the lack of methemoglobin formation in the most sensitive human group; therefore, no uncertainty factor was deemed necessary. The calculations are based on the consumption of 0.64 L of water/day by a 4-kg infant and are given in mg nitrate-nitrogen (1 mg nitrate-nitrogen = 4.4 mg nitrate). See Sects. 3.1.1.1 and 3.1.3.1 for further discussion of the principal studies. An RfD for organic nitrates as a group is not available.

3.2. INHALATION EXPOSURES

3.2.1. Acute Toxicity

3.2.1.1. Human

Atmospheric nitrates and other nitrogen oxides especially associated with smog near large industrial centers are known to contribute to the overall nitrate/nitrite intake of the population in these areas (Fan et al. 1987). However, specific information on the acute inhalation toxicity of inorganic nitrates in humans was not available.

Vasodilatory effects have been observed following inhalation exposure to various organic nitrates. Nitroglycerine and ethylene glycol dinitrate exposure (2.0 mg/m3 ethylene glycol dinitrate for 1 to 3 minutes) resulted in a drop in blood pressure and severe headaches in four out of five volunteers tested. General fatigue and pain in the chest, abdomen, and extremities were also reported. Only three out of seven volunteers experienced mild or transitory headaches at 0.5 mg/m3 ethylene glycol dinitrate (Carmichael and Lieben 1963, Stokinger 1982).

3.2.1.2. Animal

Information on the acute inhalation toxicity of inorganic nitrates in animals was not available. However, an LD50 of 1047 mg/m3 has been reported in mice for propylene glycol 1,2-dinitrate (PGDN) (Andersen and Mehl, 1973; Stokinger, 1978). In rats, however, exposure to PGDN for 4 hours to 1350 mg/m3 (200 PPM) produced no deaths or overt signs of toxicity after 14 days following treatment although the methemoglobin values increased from a mean of 6 to 23.5% (Jones et al. 1972, Stokinger 1978).

3.2.2. Subchronic Toxicity

3.2.2.1. Human

Information on the subchronic inhalation toxicity of inorganic nitrates in humans was not available. However, long-term (months to years) inhalation exposure of individuals working in munitions or dynamite manufacturing leads to an apparent biocompensation of the cardiovascular effects of organic nitrates. These workers, when removed from the source of the nitrates, experienced symptoms of angina and were subject to sudden and sometimes fatal heart attacks. This effect was documented in at least 38 dynamite workers 30-48 hours after absence from work during 1926 to 1961 (Carmichael and Lieben 1963, Stokinger 1982).

3.2.2.2. Animal

Information on subchronic inhalation toxicity of inorganic nitrates in animals was not available. Studies using monkeys, dogs, rats, and guinea pigs were performed by the U. S. Navy on the organic nitrate, propylene glycol-1,2-dinitrate (PGDN), a torpedo propellent. Animals were exposed to 0, 9, 14.5, or 31.4 ppm PGDN (0, 67, 108, and 236 mg/m3, respectively) continuously for 90 days. The animals did not exhibit visible signs of toxicity; however, methemoglobin values were increased in all species at the high dose and were highest in dogs and monkeys (23.4 and 17%, respectively). Serum inorganic nitrate was also increased to maximums of 202 µg/ml and 174 µg/ml over the control values of 12 and 2.4 µg/mL for monkeys and dogs, respectively, after 14 days of exposure to 31.4 ppm. Hemoglobin and hematocrit values were decreased 63 and 37%, respectively, in dogs. Hemosiderin deposits were observed in the liver and kidneys of dogs and rats at the high dose. Vacuolar changes associated with some iron-positive deposits, mononuclear cell infiltrates and focal necrosis were observed in the livers of all the high dose guinea pigs and in 4/9 of the high dose monkeys. Iron positive deposits were also reported in the kidneys and spleens of middle and high dose monkeys and dogs. The monkeys also had elevated serum urea nitrogen and decreased serum alkaline phosphatase levels, which could be indicative of kidney damage. Hemorrhagic foci were reported in the lungs of guinea pigs exposed to 14.5 ppm (Jones et al. 1972).

3.2.3. Chronic Toxicity

3.2.3.1. Human

Information on the chronic inhalation toxicity of inorganic nitrates in humans was not available. The effects of long-term exposure to an organic nitrate, PGDN, on tests involving eye-tracking and ataxia were performed on 115 active duty and civilian Navy personnel involved in torpedo maintenance procedures. The duration of exposures ranged from months to 11 years. The PGDN concentration during exposure ranged from near 0 to 0.22 ppm with an average concentration of 0.03 ppm. The neurological ataxia tests demonstrated no differences from the control group. When the velocity of eye movements and latency were tested immediately before and after exposure, significant decreases in eye movement velocity and latency were reported. There was no evidence; however, that any permanent neurological impairment resulted from repeated daily exposures for up to 11 years (Stokinger 1982) (see Sect. 3.2.2.1)

3.2.3.2. Animal

Information on the chronic inhalation toxicity of nitrates in animals was not available.

3.2.4. Developmental and Reproductive Toxicity

3.2.4.1. Human

Information on developmental and reproductive toxicity in humans resulting from inhalation exposure to nitrates was not available.

3.2.4.2. Animal

Information on developmental and reproductive toxicity in animals resulting from inhalation exposure to nitrates was not available.

3.2.5. Reference Concentration

3.2.5.1. Subchronic

A subchronic RfC for inhalation exposure to inorganic nitrate is not available at this time.

3.2.5.2 Chronic

A chronic RfC for inhalation exposure to inorganic nitrate is not available at this time.

3.3. OTHER ROUTES OF EXPOSURE

3.3.1. Acute Toxicity

3.3.1.1. Human

Information on the acute toxicity of inorganic nitrates in humans by other routes of exposure was unavailable. Most organic nitrates are effectively absorbed dermally or sublingually. These routes are often the most convenient route for treatment of angina. The specific efficiency of absorption varies with the particular organic moiety. For example, ethylene glycol dinitrate and nitroglycerine are readily absorbed through the skin, but erythritol tetranitrate and pentaerythritol tetranitrate are not. The cardiovascular effects particular to these compounds are essentially independent of route (Stokinger 1982) (see Sect. 3.1.1.1).

3.3.1.2. Animal

Information on the acute toxicity of inorganic nitrates in animals by other routes of exposure was unavailable. Organic nitrates are absorbed through the skin of animals, and severe acute effects have been reported. A dose of 3.5 g/kg/day PGDN applied to the backs of rabbits resulted in the death of 6 of 11 treated animals with a mean time to death of 16 days (Andersen and Mehl 1973, Stokinger 1982).

3.3.2. Subchronic Toxicity

3.3.2.1. Human

Information on the subchronic toxicity of inorganic nitrates by other routes of exposure in humans was unavailable. In the munitions and dynamite production industries, dermal absorption of organic nitrates is known to contribute to the total dose and is taken into consideration along with inhalation absorption to control worker exposure (Einert et al. 1963, Stokinger 1982) (see Sect. 3.2.2.1).

3.3.2.2 Animal

Information on the subchronic toxicity of inorganic nitrates by other routes of exposure in animals was unavailable. Doses of 1, 2, and 4 g/kg/day of propylene glycol 1,2-dinitrate were applied to the backs of 14 rabbits per group for 90 days. Weakness and slight cyanosis was observed for the first 6 days at the 2 g/kg/day dose, and one rabbit died during this period. Steady improvement in the surviving animals was seen for the remainder of the treatment period with 15% weight gain by the 20th day. In the high dose group, 13 of 14 animals died within the first 6 days. Internal organs were reported to appear dark, blue-gray (Andersen and Mehl 1973, Stokinger 1982).

3.3.3 Chronic Toxicity

3.3.3.1 Human

Information on the chronic toxicity of inorganic nitrates by other routes of exposure in animals was unavailable. Industrial exposure to organic nitrates has occurred from months to years. Dermal absorption is known to contribute to the total dose and is taken into consideration to control worker exposure (Einert et al. 1963, Stokinger 1982) (see Sect. 3.2.2.1).

3.3.3.2 Animal

Information on the chronic toxicity of nitrates by other routes of exposure in animals was unavailable.

3.3.4. Developmental and Reproductive Toxicity

3.3.4.1. Human

Information on the developmental and reproductive toxicity of nitrates by other routes of exposure in humans was unavailable.

3.3.4.2 Animal

Information on the developmental and reproductive toxicity of nitrates by other routes of exposure in humans was unavailable.

3.4. TARGET ORGANS/CRITICAL EFFECTS

3.4.1. Oral Exposures

3.4.1.1. Primary Target Organ(s)

1. Blood: formation of methemoglobinemia especially in young children. The effect depends on conversion of nitrates to nitrites in the gastrointestinal system. Methemoglobinemia is primarily caused by ingestion of inorganic nitrate but can also be the result of organic nitrate ingestion.

2. Cardiovascular system: vasodilatory effect on blood vessels. Organic nitrates are used as prophylactic agents for angina patients. Inorganic nitrate is much less effective but has also been observed to cause vasodilation, which can complicate the adverse effects of methemoglobinemia.

3.4.1.2. Other Target Organ(s)

Fetus: Studies indicate a possible fetotoxic effect at very high doses of inorganic nitrate. There are conflicting studies on this effect, which may involve the in vivo production of N-nitroso compounds.

3.4.2. Inhalation Exposures

3.4.2.1. Primary Target Organ(s)

Cardiovascular system: The vasodilatory effect of organic nitrates is independent of entry route.

3.4.2.2. Other Target Organ(s)

Blood: Methemoglobinemia can occur with organic nitrate inhalation.

3.4.3 Other Routes of Exposure

3.4.3.1 Primary target organ

Cardiovascular system: The vasodilatory effect of organic nitrates is independent of the entry route, which can include dermal or sublingual routes.

3.4.3.2 Other target organ

Blood: Methemoglobinemia can occur with organic nitrate following sublingual or dermal absorption.

4. CARCINOGENICITY

4.1. ORAL EXPOSURES

4.1.1. Human

The possibility of inorganic or organic nitrate functioning as a carcinogen depends on its conversion to nitrite and the subsequent reaction of nitrite with other molecules, specifically secondary amines, amides, and carbamates, to form carcinogenic N-nitroso compounds (Bouchard et al. 1992, Taylor and Lijinsky 1975). Human population studies have yielded conflicting results. Studies in Columbia and Allborg, Denmark, show positive correlations between the incidence of stomach cancer and the nitrate content of well water (Cuello et al. 1976, Fraser et al. 1980).

Aarhus, another town in Denmark of similar size but with relatively low nitrate concentrations in drinking water, had 25% lower stomach cancer rates in men and 20% lower rates in women (Fraser et al. 1980). Epidemiological studies in the United Kingdom have shown the overall stomach cancer deaths decreasing as the nitrate levels were rising and no positive correlation between cancer rates and areas with high nitrate in the drinking water (Forman 1985). A study of workers in a fertilizer plant exposed to high concentrations of nitrate had no significantly higher cancer rates than a control group of workers not exposed to high nitrate intakes (Al-Dabbagh et al. 1986, Croll and Hayes 1988). In an epidemiological study of cases in Ontario, Burch et al. (1987) linked diets and drinking water high in nitrate to an increased incidence of adult brain tumors. More recently, population studies in Germany failed to show any correlation between high nitrate content of drinking water and the incidence of brain tumors (Steindorf et al., 1994). The conflicting evidence reflects the complexity of the problem. Possible complications in human studies on nitrate carcinogenicity include the parameters involved in the conversion of nitrate to nitrite; the presence and concentrations of the precursor compounds to form the N-nitroso compounds; the presence and concentrations of substances that can be inhibitory to nitrosation, including vitamins C and E; and possible exogenous sources of nitrosamines (Bouchard et al. 1992).

4.1.2. Animal

Rats given both nitrite and heptamethyleneimine in drinking water, which can react in vivo to form a nitrosamine, were shown to have an increased incidence of tumors when compared to controls missing either component (Taylor and Lijinsky 1975).

4.2. INHALATION EXPOSURES

4.2.1. Human

Information on the inhalation carcinogenicity of inorganic or organic nitrate in humans was unavailable.

4.2.2. Animal

Information on the inhalation carcinogenicity of inorganic or organic nitrate in animals was unavailable.

4.3. OTHER ROUTES OF EXPOSURE

Information on the carcinogenicity of inorganic or organic nitrate in animals or humans with other routes of exposure was unavailable.

4.4. EPA WEIGHT-OF-EVIDENCE

4.4.1. Oral

CLASSIFICATION: Unavailable, currently under review (EPA 1994).

4.4.2. Inhalation

CLASSIFICATION: The carcinogenicity of nitrate in humans by the inhalation route has not been assessed.

4.5. CARCINOGENICITY SLOPE FACTORS

Carcinogenicity assessment is pending (EPA 1994).

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