This chapter summarizes the relevant epidemiologic and toxicologic studies on nitric oxide (NO). Selected chemical and physical properties, toxico-kinetic and mechanistic data, and inhalation exposure levels from the National Research Council (NRC) and other agencies are also presented. No exposure levels for NO currently exist for submarines or have been proposed by the Navy. However, exposure levels for nitrogen dioxide (NO2) have been established by the Navy and are considered to be protective against the adverse effects that might result from NO exposure. The subcommittee’s recommendations for NO exposure levels are provided at the conclusion of this chapter along with a discussion of the adequacy of the data for defining those levels and the research needed to fill the remaining data gaps.
PHYSICAL AND CHEMICAL PROPERTIES
NO is a colorless gas that combines with oxygen to form NO2 (Budavari et al. 1989). The rate of NO2 formation depends on the concentration of oxygen and the square of the concentration of NO (NIOSH 1976). At an NO concentration of 100 parts per million (ppm), NO2 forms at a rate of 2.8 ppm per minute (min) under normal atmospheric conditions (NIOSH 1976). An odor threshold of 0.3-1 ppm has been reported (ACGIH 2001). Selected physical and chemical properties are summarized in Table 9-1.
OCCURRENCE AND USE
NO is unstable in air and converts to NO2. The low-oxygen conditions on board submarines will slow NO2 formation. Because NO in air converts
TABLE 9-1 Physical and Chemical Data on Nitric Oxidea
to NO2, which is more toxic, the two chemicals should be monitored simultaneously (ACGIH 2001).
NO primarily is used as an intermediate in the production of nitric acid (Budavari et al. 1989). In recent years, NO has been recognized for its role as a regulator of cardiovascular, immune, and nervous system functions (Kiss 2000; Weinberger et al. 2001). It has been investigated and used as a treatment for various pulmonary diseases (Troncy et al. 1997).
NO is a component of smog. Sources of NO include exhaust from internal-combustion engines, smoke from fires, and tobacco smoke (ACGIH 2001). The Navy has indicated that the primary sources of nitrogen oxides on submarines are the diesel generator, the vent fog precipitator, and cigarette smoking (Crawl 2003).
SUMMARY OF TOXICITY
NO relaxes vascular smooth muscle making it an effective treatment for persistent pulmonary hypertension in newborns (Channick and Yung 1999; INO Therapeutics 2001). High NO exposures produce methemoglobinemia, a reversible event. Seger (1992) describes the clinical signs and symptoms at increasing concentrations of methemoglobin. Clinical cyanosis and “chocolate brown” blood occur in humans at about 15-20% methemo-
globin; anxiety, exertional dyspnea, weakness and fatigue, dizziness, lethargy, headache, syncope and tachycardia occur at methemoglobin concentrations between 20% and 45%; loss of consciousness begins at between 45% and 55%; and stupor, seizures, coma, bradycardia, and cardiac arrhythmias occur between 55% and 70% methemoglobin. Methemoglobin concentrations at greater than 70% lead to heart failure and death (Seger 1992).
The toxicity of air pollutants, notably NO and NO2, may be influenced by the pattern of exposure as well as concentration and duration in that cyclical peak exposures, such as those associated with rush-hour traffic, have been shown to enhance the toxic effects of NO and NO2 in animals (EPA 1993; Mercer et al. 1995). No information on pattern of exposure on submarines was provided to the subcommittee. The influence of exposure pattern on toxicity highlights the critical importance of continuous monitoring to characterize the submarine atmosphere.
Effects in Humans
Two women anesthetized with nitrous oxide and oxygen became cyanotic and developed respiratory distress (Clutton-Brock 1967). One of the women developed severe pulmonary edema and died of cardiac arrest. The other developed respiratory distress but recovered completely after oxygen and steroid therapy. Later it was discovered that the nitrous oxide cylinder was contaminated with NO, and it was estimated that the NO concentration was at least 10,000 ppm (Greenbaum et al. 1967).
Clinical studies have been conducted in patients administered therapeutic doses of NO to treat various respiratory diseases. A lung transplant patient treated with NO at 80 ppm for 8 hours (h) developed a circulating methemoglobin concentration of 9.4% (Adatia et al. 1994). When the NO concentration was reduced to 40 ppm for the ensuing 4 h, the methemoglobin concentration decreased to 6.6%. The methemoglobin concentration returned to nearly normal (0.9% increase) when the inhaled NO concentration was reduced to 20 ppm for an additional 12 h. There were no adverse health effects associated with NO treatments in patients with acute respira-
tory distress syndrome who were administered NO at 20 ppm for 48 h followed by an exposure at 10 ppm for an additional 8 days (Manktelow et al. 1997). No clinical signs were noted in patients who were treated for pulmonary hypertension and cardiac disease with NO at 40 ppm for 5 min (Pepke-Zaba et al. 1991) or in patients treated for bronchial asthma and chronic obstructive pulmonary disease with NO at 80 ppm for 10 min (Hogman et al. 1993). The methemoglobin concentration of one patient treated with NO at 80 ppm for 6 h increased to 9.4%. A second patient administered NO at 80 ppm developed a methemoglobin concentration of 14% after 18 h, and a third patient developed a concentration of 9.6% after 108 h (Wessel et al. 1994). In nine infants who had congenital heart disease and were treated 21 h postsurgery with NO at 50 ppm for 41 h, the average circulating methemoglobin concentration was 1.4%, and the NO2 concentrations were less than 2.4 ppm (Schulze-Neick et al. 1997).
Five healthy volunteers (four males and one female, ages 30-36 years) were exposed to NO at 32, 64, and 128 ppm for 3 h, and 512 ppm for 50 min (Young et al. 1994). The 512 ppm exposure was stopped when the mean methemoglobin concentration reached 5%. Six healthy male volunteers (ages 30-38 years) were exposed to NO at 100 ppm for 3 h (Young et al. 1996). It was suggested in Young et al. (1994) that maximum methemoglobin concentrations are likely reached 3-5 h after inhalation begins. Exposure to NO at up to 128 ppm for 3 h did not result in clinically significant methemoglobinemia (Young et al. 1994, 1996).
Occupational and Epidemiologic Studies
NO has been studied in association with various diseases as a component of air pollution. However, these studies often include other pollutants and lack precise measures of exposure. Further, outcomes in the general population are not relevant to the healthy submariner population. Thus, no relevant occupational and epidemiologic information was located.
Effects in Animals
Mice exposed to NO at 350 ppm for up to 8 h all died; however, exposure at 320 ppm resulted in 50% mortality, and complete survival occurred following exposure at 310 ppm (Pflesser 1935; EPA 1993). There
was no evidence of lung injury or pulmonary edema in the mice that died, and death was thought to have occurred as a result of methemoglobin formation.
Rats exposed to NO at 10 or 50 ppm for 180 min were evaluated for changes in discrimination learning using a delayed-response operant-conditioning technique (Groll-Knapp et al. 1988). The 50-ppm dose significantly decreased the number of correct trials and the total number of lever presses. Methemoglobin concentrations did not exceed 3.98%. In another study (Garat et al. 1997), no NO-related toxic effects were noted in the lungs of rats exposed to NO at 10 or 100 ppm for 40 h while breathing either 21% or 100% oxygen.
Although 11 of 20 adult male Fischer 344 rats exposed to NO at 1,000 ppm for 30 min died 30 min post-exposure, lesions were not observed in the lungs by histopathology in the rats that lived or the rats that died (Stavert and Lehnert 1990). No effects were noted in guinea pigs exposed to NO at 175 ppm for 120-150 min (Paribok and Grokholskaya 1962). No evidence of lung injury was reported in newborn lambs with persistent pulmonary hypertension when treated with NO at 80 ppm for 23 h (Zayek et al. 1993). The average methemoglobin concentration was 3%.
Anesthetized beagle dogs (3-4 per group) were exposed to NO at 0, 80, 160, 320, or 640 ppm for 6 h (Mihalko et al. 1998). One dog in the 640-ppm group died. No deaths occurred in the other groups. The circulating methemoglobin concentrations for the treatment groups were 3%, 6.6%, 24%, and 78%, respectively. Lung compliance increased during the 640-ppm exposure but remained stable in all other treatment groups. Lung resistance and peak inspiratory and expiratory flow rates were not affected. In a follow-up study to investigate potential electrocardiographic effects, conscious sling-restrained beagles were exposed to NO at 40, 80, 160, and 320 ppm via tracheal fistula. Cardiac conduction, rate, and rhythm were not affected at any concentration (Mihalko et al. 1998).
Repeated Exposures and Subchronic Toxicity
Waters et al. (1998) exposed rats by nose-only inhalation to NO at 0, 80, 200, 300, 400, or 500 ppm for 6 h per day for 1, 3, or 7 days. Exposures >300 ppm were lethal to rats after 1.5 h of exposure. Methemoglobin concentrations were elevated in groups receiving >200 ppm. Ultrastructural examination of terminal bronchioles and adjacent alveoli identified increased incidence and severity of interstitial edema in the 200-ppm group as compared with controls after 1 and 7 days of treatment. The findings
were attributed to a contaminating concentration of NO2 (2.6 ppm) produced during the exposure. In a follow-up study, rats were exposed by nose-only inhalation to NO at 0, 40, 80, 160, 200, or 250 ppm for 6 h per day for 4 weeks (Waters et al. 1998). No treatment-related effects were found by light microscopy in lung tissue or major organs. No treatment-related ultrastructural changes were observed in animals exposed at 200 ppm.
Rats exposed to NO at 0.5 ppm with two daily peak exposures at 1.5 ppm for 9 weeks showed an increased number of fenestrations in the alveolar septa of the lungs, a reduced number of interstitial cells, and a thinning of the interstitial space (Mercer et al. 1995). Although NO exposure resulted in alterations of the interstitial septa of the lungs, morphological alterations of the epithelial cells were not evident. The authors considered these lesions to be the initial stages in an emphysema-like destruction of the alveolar septa. Others have reported enlargement of air spaces in rats exposed continuously to NO at 2 ppm for 6 weeks (Azoulay et al. 1977) and many large air spaces in the periphery of the lungs of mice exposed to NO at 10 ppm for 30 weeks (Holt et al. 1979). However, similar lesions were not reported in rats exposed to NO at up to 250 ppm for 6 hrs per day for 4 weeks (Waters et al. 1998) or in mice exposed to 2.4 ppm for 23-29 months (Oda et al. 1980).
No increase in death rate was observed compared with controls in mice exposed to NO at 10 ppm for 6.5 months (Oda et al. 1976) or at 2.4 ppm NO for a lifetime (23-29 months) (Oda et al. 1980). Furthermore, the blood nitrosylhemoglobin concentration remained steady at 0.01% for lifetime exposure at 2.4 ppm and at 0.13% for 6.5-month exposure at 10 ppm. The average circulating methemoglobin concentration was 0.2% for the 6.5-month exposure, and the maximum methemoglobin concentration was 0.3% in the longer study.
No relevant information was found regarding chronic toxicity with the exception of the study noted above (Oda et al. 1980).
Reproductive Toxicity in Males
No relevant information was found regarding the potential reproductive toxicity of NO in males.
No relevant information was found regarding the potential immunotoxicity of NO.
There is a paucity of information on the genotoxicity of NO. Chromosome aberrations were not observed in Sprague-Dawley rats exposed to NO at 9, 19, or 27 ppm for 3 h (Isomura et al. 1984). A dose-related increase in the number of revertants of Salmonella typhimurium was observed in cultures exposed to atmospheres containing NO at 0-20 ppm for 30 min, but 50 ppm was cytotoxic (Arroyo et al. 1992).
No relevant information was found regarding the potential carcinogenicity of NO.
TOXICOKINETIC AND MECHANISTIC CONSIDERATIONS
NO is unstable in air and oxidizes to form NO2. However, the mechanisms of toxicity for NO and NO2 differ. NO binds to hemoglobin, resulting in reversible methemoglobinemia. NO2 exposure results in initial irritation with mild dyspnea followed by a delayed onset of pulmonary edema and, ultimately, interstitial fibrosis (Hine et al. 1970; NIOSH 1976). Thus, if an NO exposure is not sufficient to cause death, recovery can be complete. Exposures to NO2 that are not rapidly lethal might result in persistent effects and even delayed death. The conversion of NO to NO2 in medicinal applications is highly dependent on the concentration of oxygen at room temperature. Treatment of infants with NO at 50 ppm yielded NO2 concentrations <2.4 ppm (Schulze-Neick et al. 1997). Furthermore, at NO concentrations <80 ppm, there were neither significant increases in NO2 nor clinical levels of NO2 toxicity (Wessel et al. 1997; Davidson et al. 1998; Finer and Barrington 2000). In addition, exposures <100 ppm usually do not form significant amounts of methemoglobin in children or adults (Winberg et al. 1994; Roberts et al. 1997; Finer and Barrington 2000).
Inhaled NO is absorbed into the blood stream and binds to hemoglobin
to form nitrosylhemoglobin, which is rapidly oxidized to methemoglobin (Sharrock et al. 1984; Maeda et al. 1987; EPA 1993). The affinity of NO for hemoglobin is about 1,500 times greater than that of carbon monoxide (Gibson and Roughton 1957). The binding and formation of methemoglobin is NO concentration- and time-dependent (Ripple et al. 1989). The oxygen dissociation curve of methemoglobin is shifted markedly to the left, so oxygen is not easily released (Weinberger et al. 2001). Methemoglobin concentrations greater than 70% result in lethal hypoxia in humans (Seger 1992).
NO binding to hemoglobin in rats (Maeda et al. 1987), mice (Oda et al. 1980), and rabbits (Sharrock et al. 1984) is rapidly reversible and has a half-life of 15-20 min when the animals are placed in clean air. It is interesting to note that about 85-92% of NO is absorbed into the bodies of healthy humans during inhalation exposures at 0.33-5.0 ppm (Yoshida and Kasama 1987). However, only about 35% of inhaled NO is absorbed by the lungs in patients with acute lung injury who are exposed to NO at 5-40 ppm as ongoing therapy (Westfelt et al. 1997).
INHALATION EXPOSURE LEVELS FROM THE NRC AND OTHER ORGANIZATIONS
Several agencies have established or proposed inhalation exposure levels for NO. Selected values are summarized in Table 9-2.
TABLE 9-2 Selected Inhalation Exposure Levels for Nitric Oxide from Other Agenciesa
Type of Level
Exposure Level (ppm)
29 CFR 1910.1000
aThe comparability of EEGLs and CEGLs with occupational and public health standards or guidance levels is discussed in Chapter 1, section “Comparison to Other Regulatory Standards or Guidance Levels.”
Abbreviations: ACGIH, American Conference of Governmental Industrial Hygienists; NIOSH, National Institute for Occupational Safety and Health; OSHA, Occupational Safety and Health Administration; PEL, permissible exposure limit; ppm, parts per million; REL, recommended exposure limit; TLV, Threshold Limit Value; TWA, time-weighted average.
The subcommittee’s recommendations for EEGL and CEGL values for NO are summarized in Table 9-3.
Five healthy human volunteers (four males and one female, ages 30-36 years) did not manifest clinically significant methemoglobinemia when exposed to NO at 128 ppm for 3 h (average maximum methemoglobin concentration was 3.75%) (Young et al. 1994). The study suggested that maximum methemoglobin concentrations are not likely to be reached until 3-5 h of exposure. On the basis of Young et al. (1994), the subcommittee recommends a 1-h EEGL value of 130 ppm. Because Young et al. (1994) employed healthy human subjects, no uncertainty factors were applied. The recommended 1-h EEGL is supported by studies in rats and dogs demonstrating an absence of effects on methemoglobin concentrations and lung or cardiac functions during exposures at 200 ppm or less for 6 h (Mihalko et al. 1998; Waters et al. 1998).
At about 15-20% methemoglobin, clinical cyanosis, and “chocolate brown” blood begin to appear (Seger 1992). Thus, an increase in methemo-
TABLE 9-3 Emergency and Continuous Exposure Guidance Levels for Nitric Oxide (ppm)a
globin formation of 10-15% could be used as a point of departure for adverse health effects resulting from NO exposure. In three patients treated for respiratory disease with NO at 80 ppm, methemoglobin concentrations were 9.6% after 108 h exposure, 14% after 18 h, and 9.4% after 6 h (Wessel et al. 1994). When the 80-ppm exposure was decreased, the percent of methemoglobinemia also decreased. In another study, a lung transplant patient treated with NO at 80 ppm for 8 h developed a circulating methemoglobin concentration of 9.4% (Adatia et al. 1994). Average methemoglobin concentrations were 1.4% in infant patients exposed to NO at 50 ppm for a mean of 41 h (Schulze-Neick et al. 1997). In healthy subjects exposed at 128 ppm for 3 h there was no clinical evidence of significant methemoglobinemia (the average methemoglobin concentration was 3.75%) (Young et al. 1994). On the basis of this information in both healthy adults and sensitive patient populations, exposures to NO at 50 ppm for 24 h would not be expected to cause any adverse health effects in a noncompromised adult human population.
No long-term human exposure studies that used sufficiently high concentrations of NO to identify a no-observed-adverse-effect level (NOAEL) or lowest-observed-adverse-effect level (LOAEL) for disease or lethality were found. However, in mice exposed to NO at 10 ppm for 6.5 months or at 2.4 ppm for 23-29 months, the average methemoglobin concentration was 0.2% at 10 ppm, and the maximum methemoglobin concentration was 0.3% at 2.4 ppm. There were no significant signs of disease or difference in death rates compared with controls (Oda et al. 1976; Oda et al. 1980). The subcommittee selected 10 ppm as the NOAEL to develop a 90-day CEGL. Applying an interspecies uncertainty factor of 3, the 90-day CEGL is 3 ppm. Because there is minimal variation in methemoglobin concentrations in the animal data and in the human data available for shorter durations, no additional uncertainty factors were applied.
DATA ADEQUACY AND RESEARCH NEEDS
Sufficient data are available to develop 1-h exposure limits. Additional nonlethal exposure data would assist in deriving 24-h exposure limits, because the present recommendations primarily rely on limited data in
respiratory-compromised patients who might be less sensitive to NO than normal humans. There are no supporting long-term studies on NO available to determine 90-day exposure limits or to determine the carcinogenicity of NO. Thus, well-designed, continuous 90-day and lifetime studies would provide needed information to develop 90-day exposure limits and to determine the carcinogenic potential of NO.
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