1 NAC/Interim (NO2)/Proposed (N2O4): December 2008
2
3
4
5
6
7
8
9
10 ACUTE EXPOSURE GUIDELINE LEVELS (AEGLs)
11
12 FOR
13
14 NITROGEN DIOXIDE
15 (CAS Reg. No. 10102-44-0)
16
17 NITROGEN TETROXIDE
is (CAS Reg. No. 10544-72-6)
19
20
21
22
23
24
25
26 INTERIM/PROPOSED
27
28
29
30
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 PREFACE
2
3 Under the authority of the Federal Advisory Committee Act (FACA) P. L. 92-463
4 of 1972, the National Advisory Committee for Acute Exposure Guideline Levels for
5 Hazardous Substances (NAC/AEGL Committee) has been established to identify, review
6 and interpret relevant toxicologic and other scientific data and develop AEGLs for high
7 priority, acutely toxic chemicals.
8
9 AEGLs represent threshold exposure limits for the general public and are
10 applicable to emergency exposure periods ranging from 10 minutes to 8 hours. Three
11 levels— AEGL-1, AEGL-2 and AEGL-3— are developed for each of five exposure
12 periods (10 and 30 minutes, 1 hour, 4 hours, and 8 hours) and are distinguished by
13 varying degrees of severity of toxic effects. The three AEGLs are defined as follows:
14
15 AEGL-1 is the airborne concentration (expressed as parts per million or
16 milligrams per cubic meter [ppm or mg/m3]) of a substance above which it is predicted
17 that the general population, including susceptible individuals, could experience notable
18 discomfort, irritation, or certain asymptomatic, non-sensory effects. However, the effects
19 are not disabling and are transient and reversible upon cessation of exposure.
20
21 AEGL-2 is the airborne concentration (expressed as ppm or mg/m3) of a
22 substance above which it is predicted that the general population, including susceptible
23 individuals, could experience irreversible or other serious, long-lasting adverse health
24 effects or an impaired ability to escape.
25
26 AEGL-3 is the airborne concentration (expressed as ppm or mg/m3) of a
27 substance above which it is predicted that the general population, including susceptible
28 individuals, could experience life-threatening health effects or death.
29
30 Airborne concentrations below the AEGL-1 represent exposure levels that can
31 produce mild and progressively increasing but transient and nondisabling odor, taste, and
32 sensory irritation, or certain asymptomatic, non-sensory effects. With increasing airborne
33 concentrations above each AEGL, there is a progressive increase in the likelihood of
34 occurrence and the severity of effects described for each corresponding AEGL. Although
35 the AEGL values represent threshold levels for the general public, including susceptible
36 subpopulations, such as infants, children, the elderly, persons with asthma, and those with
37 other illnesses, it is recognized that individuals, subject to unique or idiosyncratic
38 responses, could experience the effects described at concentrations below the
39 corresponding AEGL.
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 TABLE OF CONTENTS
2
3 LIST OF TABLES 5
4 LIST OF FIGURES 5
5 SUMMARY 6
6 1. INTRODUCTION 10
7 2. HUMAN TOXICITY DATA 12
8 2.1. Acute Lethality 12
9 2.2. Nonlethal Toxicity 13
10 2.2.1. Case Reports 13
11 2.2.2. Epidemiologic Studies 15
12 2.2.3. Experimental Studies 17
13 2.3. Developmental/Reproductive Toxicity 21
14 2.4. Genotoxicity 22
15 2.5. Carcinogenicity 22
16 2.6. Summary 22
17 3. ANIMAL TOXICITY DATA 22
18 3.1. Acute Lethality 22
19 3.1.1. Dogs 23
20 3.1.2. Rabbits 23
21 3.1.3. Guinea Pigs 24
22 3.1.4. Rats 25
23 3.1.5. Mice 26
24 3.2. Nonlethal Toxicity 27
25 3.2.1. Monkeys 27
26 3.2.2. Dogs 27
27 3.2.3. Rabbits 28
28 3.2.4. Sheep 29
29 3.2.5. Guinea Pigs 29
30 3.2.6. Hamsters 30
31 3.2.7. Ferrets 30
32 3.2.8. Rats 31
33 3.2.9. Mice 34
34 3.3. Developmental/Reproductive Toxicity 35
35 3.4. Genotoxicity 35
36 3.5. Subchronic and Chronic Toxicity/Carcinogenicity 35
37 3.6. Summary 36
38 4. SPECIAL CONSIDERATIONS 36
39 4.1. Metabolism and Disposition 36
40 4.2. Mechanism of Toxicity 37
41 4.3. Oxides of Nitrogen 38
42 4.4. Other Relevant Information 39
43 4.4.1. Species Variability 39
44 4.4.2. Susceptible Populations 39
45 4.4.3. Concentration-Response Relationship 40
46 4.4.4. Susceptibility to infection 41
47 5. DATA ANALYSIS FOR AEGL-1 42
48 5.1. Summary of Human Data Relevant to AEGL-1 42
49 5.2. Summary of Animal Data Relevant to AEGL-1 43
50 5.3. Derivation of AEGL-1 43
51 6. DATA ANALYSIS FOR AEGL-2 43
52 6.1. Summary of Human Data Relevant to AEGL-2 44
53 6.2. Summary of Animal Data Relevant to AEGL-2 44
54 6.3. Derivation of AEGL-2 45
55 7. DATA ANALYSIS FOR AEGL-3 45
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 7.1. Summary of Human Data Relevant to AEGL-3 45
2 7.2. Summary of Animal Data Relevant to AEGL-3 46
3 7.3. Derivation of AEGL-3 46
4 8. SUMMARY OF AEGLS 47
5 8.1. AEGL Values and Toxicity Endpoints 47
6 8.2. Comparison with Other Standards and Criteria 48
7 9. REFERENCES 51
8 APPENDIX A: Derivation of AEGL Values 64
9 APPENDIX B: Derivation Summary for AEGL Values for Nitrogen Oxides 68
10 APPENDIX C: Time Scaling Category Plot for Nitrogen Oxides 72
11
12
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 LIST OF TABLES
2
3 SI: Summary of AEGL Values for Nitrogen Dioxide 9
4 S 2: Summary of AEGL Values for Nitrogen Tetroxide 9
5
6 TABLE LPHYSICOCHEMICAL DATA FOR NITROGEN DIOXIDE 11
7 TABLE 2: PHYSICOCHEMICAL DATA FOR NITROGEN TETROXIDE 12
8 TABLE 3: Effects of acute exposure to high NO2 concentrations 13
9 TABLE 4: Summary of NO2 Mortality for Five Species 27
10 TABLES: AEGL-IValues for Nitrogen Dioxide and Nitrogen Tetroxide 43
11 TABLE 6: AEGL-2 Values for Nitrogen Dioxide and Nitrogen Tetroxide 45
12 TABLE 7: AEGL-3 Values for Nitrogen Dioxide and Nitrogen Tetroxide 47
13 TABLES: Summary of AEGL Values for Nitrogen Dioxide (mg/m3 [ppm]) 47
14 TABLE 9: Summary of AEGL Values for Nitrogen Tetroxide (mg/m3 [ppm]) 48
15 TABLE 10: Extant Standards and Guidelines for Nitrogen Dioxide 49
16
17
18 LIST OF FIGURES
19
20 Figure 1: Category plot of AEGL values and effects of nitrogen dioxide on humans and animals 73
21
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 SUMMARY
2
3 Nitrogen oxide compounds occur from both natural and anthropogenic sources.
4 Nitrogen dioxide (NO2) is the most ubiquitous of the oxides of nitrogen and has the
5 greatest impact on human health. Nitrogen tetroxide (TSPzO^ is a component of rocket
6 fuels. NC>2 exists as an equilibrium mixture of NC>2 and N2O4 but the dimer is not
7 important at ambient concentrations (U.S. EPA 1993). The two compounds are phase-
8 related forms with N2O4 favored in the liquid phase and NO2 favored in the gaseous
9 phase. As a result when ^64 is released it vaporizes and dissociates into NO2, making
10 it nearly impossible to generate a significant concentration of N2©4 at atmospheric
11 pressure and ambient temperatures, without generating a vastly higher concentration of
12 NO2. Very few inhalation toxicity data are available on N2O4. Thus, the AEGL values
13 were developed based on data for NC>2, the predominant form, and values are considered
14 applicable to all nitrogen oxides. Values for N2O4 in units of ppm have been calculated
15 on a molar basis as presented below.
16
17 NC>2 is an irritant to the mucous membranes and may cause coughing and dyspnea
18 during exposure. After less severe exposure, symptoms may persist for several hours
19 before subsiding (NIOSH 1976). With more severe exposure, pulmonary edema ensues
20 with signs of chest pain, cough, dyspnea, cyanosis, and moist rales heard on auscultation
21 (NIOSH 1976, Douglas et al. 1989). Death from NO2 inhalation is caused by
22 bronchospasm and pulmonary edema in association with hypoxemia and respiratory
23 acidosis, metabolic acidosis, shift of the oxyhemoglobin dissociation curve to the left,
24 and arterial hypotension (Douglas et al. 1989). A characteristic of NO2 intoxication after
25 the acute phase is a period of apparent recovery followed by late-onset bronchiolar injury
26 that manifests as bronchiolitis fibrosa obliterans (NIOSH 1976, NRC 1977, Hamilton
27 1983, Douglas et al. 1989). In addition, experiments with laboratory animals indicate
28 that exposure to NO2 increases susceptibility to infection (Henry et al. 1969, U.S. EPA
29 1993) due, in part, to alterations in host pulmonary defense mechanisms (Gardner et al.
30 1969).
31
32 For AEGL-1 a concentration of 0.5 ppm was adopted for all time points.
33 Although the response of asthmatics to NO2 is variable, asthmatics were identified as a
34 potentially susceptible population. The evidence indicates that some asthmatics exposed
35 to 0.3-0.5 ppm NO2 may respond with either subjective symptoms or slight changes in
36 pulmonary function of no clinical significance. In contrast, some asthmatics did not
37 respond to NO2 at concentrations of 0.5-4 ppm. Because of the weight of evidence, the
38 study by Kerr et al. (1978, 1979) was considered the most appropriate for derivation of
39 AEGL-1 values. They reported that 7/13 asthmatics experienced slight burning of the
40 eyes, slight headache, and chest tightness or labored breathing with exercise during
41 exposure to 0.5 ppm for 2 hours; at this concentration the odor of NO2 was perceptible
42 but the subjects became unaware of it after about 15 minutes. No changes in any
43 pulmonary function tests were found immediately following the chamber exposure (Kerr
44 et al. 1978, 1979). Therefore, 0.50 ppm was considered a no-adverse-effect level for the
45 asthmatic population. Since asthmatics are potentially the most susceptible population,
46 no uncertainty factor was applied. Time scaling was not done because adaptation to mild
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 sensory irritation occurs. In addition, animal responses to NC>2 exposure have
2 demonstrated a much greater dependence upon concentration than upon time; therefore,
3 extending the 2-hour concentration to 8 hours should not exacerbate the human response.
4
5 Supporting studies for AEGL-1 report findings similar to the key studies.
6 Significant group mean reductions in FEVi (-17.3% with NC>2 vs -10.0% with air) and
7 specific airway conductance (-13.5% with NC>2 vs -8.5% with air) occurred in asthmatics
8 after exercise during exposure to 0.3 ppm for 4 hours and 1/6 individuals experienced
9 chest tightness and wheezing (Bauer et al. 1985). The onset of effects was delayed when
10 exposures were by oral-nasal inhalation as compared to oral inhalation and may have
11 resulted from scrubbing within the upper airway. In a similar study, asthmatics exposed
12 to 0.3 ppm for 30 minutes at rest followed by 10 minutes of exercise had significantly
13 greater reductions in FEVi (10% vs 4% with air) and partial expiratory flow rates at 60%
14 of total lung capacity, but no symptoms were reported (Bauer et al. 1986). In a
15 preliminary study with 13 asthmatics exposed to 0.3 ppm for 110 minutes, slight cough
16 and dry mouth and throat and significantly greater reduction in FEVi occurred after
17 exercise (11% vs 7%); however, in a larger study, no changes in pulmonary function
18 were measured and no symptoms were reported following exposure of 21 asthmatics to
19 concentrations up to 0.6 ppm for 75 minutes (Roger et al. 1990).
20
21 Human data were also used as the basis for AEGL-2. Three healthy male
22 volunteers experienced definite discomfort from exposure to 30 ppm for 2 hours
23 (Henschler et al. 1960). Three individuals exposed to 30 ppm for 2 hours perceived an
24 intense odor upon entering the chamber, but the odor perception quickly diminished and
25 was completely absent after 25-40 minutes. One individual experienced a slight tickling
26 of the nose and throat mucous membranes after 30 minutes, the two others after 40
27 minutes. From 70 minutes on, all subjects experienced a burning sensation and an
28 increasingly severe cough for the next 10-20 minutes, but coughing decreased from 100
29 minutes on. However, the burning sensation continued and moved into the lower
30 sections of the airways and was finally felt deep in the chest. At this time, marked
31 sputum secretion and dyspnea were noted. Toward the end of the exposure, the subjects
32 reported the exposure conditions to be bothersome and barely tolerable. A sensation of
33 pressure and increased sputum secretion continued for several hours after cessation of
34 exposure (Henschler et al. 1960). The point of departure is considered a threshold for
35 AEGL-2 since the effects noted by the subjects would not impair the ability to escape and
36 the effects were reversible after cessation of exposure.
37
38 AEGL-3 values were based on animal data and supported by a human case report.
39 Exposure of monkeys to 50 ppm for 2 hours was used to derive the AEGL-3 values.
40 Monkeys (n = 2-6/group) were exposed to 10-50 ppm NO2 for 2 hours with respiratory
41 function monitored during exposure (Henry et al. 1969). NO2 exposure alone resulted in
42 a markedly increased respiratory rate and decreased tidal volume during exposures to 50
43 and 35 ppm, but only slight effects at 15 and 10 ppm. Mild histopathological changes in
44 the lungs were noted after exposure to 10 and 15 ppm, however, marked changes in lung
45 structure were observed after exposure to 35 and 50 ppm. The alveoli were expanded
46 with septal wall thinning, bronchi were inflamed with proliferation or erosion of the
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 surface epithelium, and lymphocyte infiltration was seen with edema. In addition to the
2 effects on the lungs, interstitial fibrosis (35 ppm) and edema (50 ppm) of cardiac tissue,
3 glomerular tuft swelling in the kidney (35 and 50 ppm), lymphocyte infiltration in the
4 kidney and liver (50 ppm), and congestion and centrilobular necrosis in the liver (50
5 ppm) were observed.
6
7 The AEGL-3 values are supported by human data for a welder. Pulmonary
8 edema, confirmed on X-ray and requiring medical intervention, resulted from exposure to
9 approximately 90 ppm for up to 40 minutes (Norwood et al. 1966). If this exposure
10 scenario is used for derivation of AEGL-3 values with an uncertainty factor of 3 the
11 values are nearly identical to those derived using the data in the monkey. In addition, the
12 AEGL-3 values are below the concentrations at which lethality first occurred in five
13 animal species: 75 ppm for 4 hours in the dog and 1 hour in the rabbit, 50 ppm for 1 hour
14 in the guinea pig, and 50 ppm for 24 hours in the rat and mouse (Hine et al. 1970).
15
16 For AEGL-2 and AEGL-3, the 10- and 30-minute, and 1-, 4-, and 8-hour AEGL
17 endpoints were calculated from Cn x t = k using n = 3.5 (ten Berge et al. 1986). The
18 value of n was calculated by ten Berge et al. using the data of Hine et al. (1970) in five
19 species of laboratory animal. A total uncertainty factor of 3 was applied which includes a
20 3 for intraspecies variability and a 1 for interspecies variability. Use of a greater
21 intraspecies uncertainty factor was not considered necessary because the mechanism of
22 action is not expected to differ greatly among individuals. Because human data were
23 used as the point of departure for AEGL-2, the endpoint in the monkey study is below the
24 definition of AEGL-3, human data support the AEGL-3 point of departure and derived
25 values, the mechanism of action does not vary between species with the target at the
26 alveoli, and due to the similarities of the respiratory tract between humans and monkeys,
27 additional interspecies uncertainty factors are not considered necessary.
28
29 The values for the three AEGL classifications for the five time periods are listed
30 in Table S 1 for NO2 and Table S 2 for N2O4.
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S 1: Summary of AEGL Values for Nitrogen Dioxide
Classification
AEGL-lb
(Non-
disabling)
AEGL-2
(Disabling)
AEGL-3
(Lethal)
10-Minute
0.94
mg/m3
(0.50 ppm)
38 mg/m3
(20 ppm)
64 mg/m3
(34 ppm)
30-Minute
0.94
mg/m3
(0.50 ppm)
28 mg/m3
(15 ppm)
47 mg/m3
(25 ppm)
1-Hour
0.94
mg/m3
(0.50 ppm)
23 mg/m3
(12 ppm)
38 mg/m3
(20 ppm)
4-Hour
0.94
mg/m3
(0.50 ppm)
15 mg/m3
(8.2 ppm)
26 mg/m3
(14 ppm)
8-Hour
0.94
mg/m3
(0.50 ppm)
13 mg/m3
(6.7 ppm)
21 mg/m3
(11 ppm)
Endpoint" (Reference)
slight burning of the eyes,
slight headache, chest
tightness or labored
breathing with exercise in
7/13 asthmatics (Kerr et
al. 1978, 1979)
burning sensation in nose
and chest, cough,
dyspnea, sputum
production in normal
volunteers (Henschler et
al. 1960)
marked irritation;
histopath in lungs; fibrosis
and edema of cardiac
tissue; necrosis in liver;
no deaths in monkeys
(Henry etal. 1969)
aSome effects may be delayed.
bThe sweet odor of NO2 may be perceptible to most individuals at this concentration; however, adaptation
occurs rapidly.
S 2: Summary of AEGL Values for Nitrogen Tetroxide
Classification
AEGL-lb
(Non-
disabling)
AEGL-2
(Disabling)
AEGL-3
(Lethal)
10-Minute
0.94
mg/m3
(0.25 ppm)
38 mg/m3
(10 ppm)
64 mg/m3
(17 ppm)
30-Minute
0.94
mg/m3
(0.25 ppm)
28 mg/m3
(7.6 ppm)
47 mg/m3
(13 ppm)
1-Hour
0.94
mg/m3
(0.25 ppm)
23 mg/m3
(6.2 ppm)
38 mg/m3
(10 ppm)
4-Hour
0.94
mg/m3
(0.25 ppm)
15 mg/m3
(4.1 ppm)
26 mg/m3
(7.0 ppm)
8-Hour
0.94
mg/m3
(0.25 ppm)
13 mg/m3
(3.5 ppm)
21 mg/m3
(5.7 ppm)
Endpoint" (Reference)
slight burning of the eyes,
slight headache, chest
tightness or labored
breathing with exercise in
7/13 asthmatics (Kerr et
al. 1978, 1979)
burning sensation in nose
and chest, cough,
dyspnea, sputum
production in normal
volunteers (Henschler et
al. 1960)
marked irritation;
histopath in lungs; fibrosis
and edema of cardiac
tissue; necrosis in liver;
no deaths in monkeys
(Henry etal. 1969)
9
10
11
aSome effects may be delayed.
bThe sweet odor of NO2 may be perceptible to most individuals at this concentration; however, adaptation
occurs rapidly.
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 1. INTRODUCTION
2
3 Nitrogen dioxide (NO2) is the most ubiquitous of the oxides of nitrogen and has
4 the greatest impact on human health. NO2, which exists as an equilibrium mixture of
5 NO2 and ^64 (nitrogen tetroxide), is a reddish-brown gas with a sweet odor, is heavier
6 than air, and reacts with water (U.S. EPA 1993, Mohsenin 1994). NC>2 is shipped under
7 pressure and the equilibrium between NO2 and N2O4 is altered with change in pressure
8 with N2O4 becoming predominant at very high pressures. NO2 is a free radical with
9 sufficient stability to exist in relatively high concentrations in ambient air (Mohsenin
10 1994).
11
12 The major source of atmospheric NC>2 is from the combustion of fossil fuels for
13 heating, household appliances, power generation, and in motor vehicles. Consequently,
14 the chemical is a major contributor to smog and a concern for indoor air quality.
15 Ambient levels in urban air pollution episodes in the US have been measured between 0.1
16 and 0.8 ppm as a maximum hourly average with short-term peaks as high as 1.27 ppm.
17 Indoor NO2 concentrations may reach a maximum 1 hour level of 0.25 to 1.0 ppm with
18 peak levels as high as 2-4 ppm where gas appliances or kerosene heaters are used
19 (Mohsenin 1994).
20
21 N2O4 is a commonly used as a rocket propellant (Yue et al. 2004). Limited
22 toxicity data on N2O4 show effects similar to those of NC>2.
23
24 No toxicity data or information on the uses or sources of nitrogen trioxide (N2O3)
25 were found. Information on the chemical interactions of N2Os with the other oxides of
26 nitrogen was not available. Therefore, N2Os will not be considered further here.
27
28 NC>2 is an irritant to the mucous membranes and may cause coughing and dyspnea
29 during exposure. After less severe exposure, symptoms may persist for several hours
30 before subsiding (NIOSH 1976). With more severe exposure, pulmonary edema ensues
31 with chest pain, cough, dyspnea, cyanosis, and moist rales heard on auscultation (NIOSH
32 1976, Douglas et al. 1989). Death from NO2 inhalation is caused by bronchospasm and
33 pulmonary edema in association with hypoxemia and respiratory acidosis, metabolic
34 acidosis, shift of the oxyhemoglobin dissociation curve to the left, and arterial
35 hypotension (Douglas et al. 1989). A characteristic of NO2 intoxication after the acute
36 phase is a period of apparent recovery followed by late-onset bronchiolar injury that
37 manifests as bronchiolitis fibrosa obliterans (NIOSH 1976, NRC 1977, Hamilton 1983,
38 Douglas etal. 1989).
39
40 Selected physicochemical properties of nitrogen dioxide and of nitrogen tetroxide
41 are listed in Tables 1 and 2, respectively.
42
43
10
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TABLE 1: PHYSIC OCHEMICAL DATA FOR NITROGEN DIOXIDE
Parameter
Common name
Synonyms
CAS registry no.
Chemical formula
Molecular weight
Physical state
Vapor pressure
Vapor density (air =1)
Melting/boiling point
Flamability
Solubility in water
Conversion factors in air
Reactivity
Value
nitrogen dioxide
10102-44-0
NO2
46.01
reddish-brown gas
720 torr at 20°C; 800 mm Hg at
25°C
1.58
-9.3°C/21.15°C
does not burn
0.037 niL/mL at 35°C
1 ppm = 1.88 mg/m3
1 mg/m3 = 0.53 ppm
decomposes in water forming nitric
oxide and nitric acid
Reference
Budavari et al. 1996
Budavari et al. 1996
Budavari et al. 1996
ACGIH 1991, U.S. EPA 1990
Budavari et al. 1996
Budavari et al. 1996
Budavari et al. 1996
Mohsenin 1994
U.S. EPA 1993
Budavari et al. 1996
11
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TABLE 2: PHYSICOCHEMICAL DATA FOR NITROGEN TETROXIDE
Parameter
Common name
Synonyms
CAS registry no.
Chemical formula
Molecular weight
Physical state
Vapor pressure
Vapor density (air = 1)
Melting/boiling point
Flamability
Solubility in water
Conversion factors in air
Reactivity
Value
dinitrogen dioxide
10544-72-6
N2O4
92.01
colored liquid
760mmHgat21°C
1.45at20°C
-9.3°C/21.5°C
No data
No data
1 ppm= 3.70 mg/m3
1 mg/m3 = 0.27 ppm
Reacts violently with organic
compounds; reacts with water
Reference
Lide 1988
Lide 1988
Lide 1988
Lide 1988
Lide 1988
Kushneva and Gorshkova 1999,
Lide 1988
calculated
Kushneva and Gorshkova 1999,
Lide 1988
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
2. HUMAN TOXICITY DATA
2.1. Acute Lethality
Book (1982) used allometric scaling based on minute volume and LCso values for
NC>2 for 5 animal species to calculate a human 1-hour LCso of 174 ppm. Concentrations
of >200 ppm are reported to induce immediate symptoms of bronchospasm and
pulmonary edema and may cause syncope, unconsciousness, and quick death (Douglas et
al. 1989).
Clinical responses to "acute" inhalation of high concentrations of NC>2 based on
occupational exposures, are given in Table 3 (NRC 1977). Durations of exposures were
not specified except for the statement that workers in a nitric acid manufacturing plant in
Italy were exposed to average concentrations of 30-35 ppm for an unspecified number of
years with no adverse signs or symptoms.
12
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TABLE 3: Effects of acute exposure to high NO2 concentrations
Concentration (ppm)
0.4
15-25
25-75
150-300+
Effect
approximate odor threshold
respiratory and nasal irritation
reversible pneumonia and bronchiolitis
fatal bronchiolitis and bronchopneumonia
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
From NRC 1977.
2.2. Nonlethal Toxicity
2.2.1. Case Reports
Probably the most well known occupational manifestation of NO2 toxicity is that
of silo filler's disease. In a silo, the gas that accumulates above the silage is depleted of
oxygen, is rich in carbon dioxide, and contains a mixture of nitrogen oxides, mainly
NC>2 which can reach concentrations of 200 to 4000 ppm within 2 days (Lowry and
Schuman 1956, Douglas et al. 1989). The term silo filler's disease was first used by
Lowry and Schuman in 1956 in an article in which they described the clinical
progression of the disease: inhalation of irritant gas from a silo; immediate cough and
dyspnea with a sensation of choking; 2-3 week period after exposure of apparent
remission; second phase of illness accompanied by fever with progressively more severe
dyspnea, cyanosis, and cough; inspiratory and expiratory rales; discrete nodular
densities on the lung; and neutrophilic leukocytosis (Lowry and Schuman 1956).
Douglas et al. (1989) reported on 17 patients examined at the Mayo clinic between 1955
and 1987 after exposure to silo gas. Eye irritation was described during exposure, acute
lung injury occurred in 11 individuals, and 16 had either persistent or delayed symptoms
of dyspnea, cough, chest pain, and rapid breathing. One patient died and at autopsy
diffuse alveolar damage with hyaline membranes and hemorrhagic pulmonary edema
and acute edema of the airways were observed. Bronchiolitis fibrosa obliterans
developed in one patient many years later; however, prophylactic administration of
corticosteroids may have prevented chronic obstructive pulmonary disease (COPD) in
the other patients. Similar case reports and outcomes of silo filler's disease and
industrial exposure were described in earlier literature (Grayson 1956, Lowry and
Schuman 1956, Milne 1969).
A welder developed shortness of breath and chest discomfort during the use of an
acetylene torch for metal-cutting in a poorly ventilated water main; the worker had spent
approximately 30 minutes welding in the confined space before being forced to vacate.
Several hours later, the worker became so short of breath that he couldn't sleep that night.
Chest X-ray 18 hours after exposure revealed pulmonary edema and a pulmonary
function test showed 42% of the predicted value for FVC. The individual was admitted
to the hospital and treated with antibiotics and oxygen. The patient fully recovered by 21
days after exposure. Simulation of the accident produced an NO2 concentration of 90
ppm within 40 minutes and total oxides of nitrogen in excess of 300 ppm (Norwood et al.
13
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 1966). It can be assumed that the individual was exposed to at least 90 ppm during the
2 welding operation and that the outcome could have been more severe, or even fatal,
3 without medical intervention.
4
5 An outbreak of NO2-induced respiratory illness was reported among players and
6 spectators at two high school hockey games (Hedberg et al. 1989). Patients presented
7 with acute onset of cough, hemoptysis, and/or dyspnea during or within 48 hours of
8 attending the hockey game. No changes in lung function were measured 10 days and 2
9 months after exposure. NO2 concentrations were not measured in the arena during the
10 outbreak, but the source was traced to a malfunctioning motor on the ice resurfacer.
11 Other cases of respiratory illness in hockey players, referees, and spectators have been
12 associated with elevated nitrogen dioxide levels in the arena due to malfunctioning
13 resurfacers or ventilation systems combined with elevated carbon monoxide levels (Smith
14 et al. 1992, Soparkar et al. 1993, Morgan 1995, Karl son-Stiber et al. 1996). Attempts to
15 measure NO2 concentrations in the arenas or reconstruct the situations were described by
16 the authors as not indicative of the actual exposure scenario which resulted in adverse
17 effects.
18
19 Morley and Silk (1970) described a number of cases in which welders involved in
20 ship repair and shipbuilding were exposed to nitrous fumes. Symptoms included
21 dyspnea, cough, headache, tightness/pain in chest, nausea, and cyanosis. Most patients
22 recovered after treatment with oxygen and antibiotics; however, one man died 43 days
23 later from viral pneumonia. Two individuals admitted to the hospital with cyanosis,
24 dyspnea, and pulmonary edema, were exposed to a concentration of NO2 measured at 30
25 ppm during a 40-minute welding operation. However, the authors noted that 7 other
26 individuals present at the time were unaffected.
27
28 A railroad tank car ruptured at a chemical plant releasing a cloud of NO2 in a
29 small community (Bauer et al. 1998). In the first 30 hours after the release, the most
30 common symptoms reported in emergency room visits were headache, burning eyes, and
31 sore throat. Most air samples collected 3-7 hours after the release showed concentrations
32 of 0 ppm with one sample showing 1.4 ppm. No attempt was made to correlate
33 symptoms with estimated exposure.
34
35 Acute toxic reactions were described in four fireman who were exposed to NO2
36 which originated from a leak in a chemical plant (Tse and Bockman 1970).
37 Concentrations were not reported and exposure durations were defined as "barely a few
38 minutes" to "about ten minutes." Initial responses, which cleared within several days,
39 included headache, a dry hacking cough, pulmonary edema, sinusitis, and/or upper
40 respiratory tract irritation. Four to six weeks after exposure, three of the patients
41 developed fever, chest tightness, shortness of breath, and a productive cough; these
42 subsided and the patients remained asymptomatic. The fourth patient developed
43 chronic pulmonary insufficiency, consisting of dyspnea on exertion, despite normal
44 chest X-ray.
45
46 A large number of patients were treated for respiratory complaints following
14
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 release of a cloud of ^64 from a railroad tank car. The most common symptoms were
2 headache, burning eyes, and sore throat; an abnormal lung exam and an abnormal chest
3 x-ray were also reported for some individuals, but these findings were not further defined
4 (Bauer et al. 1996). No pulmonary edema or deaths were attributed to the accident.
5 However, six individuals were later diagnosed with reactive airways dysfunction
6 syndrome (RADS) three months after exposure (Conrad et al. 1998). Concentrations of
7 various oxides of nitrogen in the cloud were not reported.
8
9 Four cases of exposure to unknown concentrations of nitrous fumes were reported
10 for individuals involved in either the use of an oxyacetylene burner during a leak at a
11 chemical plant, or in shotfiring (Jones et al. 1973). Three patients presented with
12 pulmonary edema, one of which progressed to bronchiolitis obliterans; the fourth patient
13 presented with clinical features of bronchi olitis obliterans. All recovered completely
14 following corticosteroid treatment.
15
16 2.2.2. Epidemiologic Studies
17
18 Several epidemiological studies associating ambient NC>2 exposure with an
19 increase in the prevalence of respiratory illness have been inconclusive. Increased odds
20 ratios (1.2-1.7) were found for bronchitis, chronic cough, and chest illness but not for
21 wheeze and asthma in children from six U.S. cities with annual average NC>2 levels
22 0.0065-0.0226 ppm (Dockery et al. 1989). No association was found between long-term
23 differences in NC>2 levels (change of 0.0106 ppm/6-week average) and mean annual rates
24 of respiratory episodes in children from urban and rural regions in Switzerland, however
25 the duration of symptoms was increased (Braun-Fahrlaender et al. 1992). An increase in
26 the cases of croup in children was associated with total suspended particulate matter and
27 NC>2 (Schwartz et al. 1991) and decreased lung function in children was linked to sulfur
28 dioxide in combination with NO2 (Mostardi et al. 1981). Symptoms of chronic
29 obstructive pulmonary disease have been linked to exposure to total oxidants (>0.1 ppm),
30 NO2, and/or sulfates, but not to NO2 alone (Detels et al. 1981, Euler et al. 1988).
31 Combined effects of NC>2, SC>2, particulate matter, H^S, and other pollutants were
32 considered as contributing factors to a positive association between the occurrence of
33 upper respiratory infections and living in polluted areas of Finland for children <2 years
34 and 6 years old (Jaakkola et al. 1991).
35
36 In a more recent study, children from 12 communities in California were assessed
37 for respiratory disease prevalence and pulmonary function (Peters et al. 1999a,b).
38 Wheeze prevalence was correlated with levels of both acid and NC>2 in boys, whereas
39 regression analysis showed that NC>2 was significantly associated with lower FVC, FEVi,
40 and maximal midexpiratory flow in girls. When these data were further analyzed by
41 month (Millstein et al. 2004), wheezing during the spring and summer months was not
42 associated with either nitric acid or NC>2. However among asthmatics, the monthly
43 prevalence of asthma medication use was associated with monthly levels of ozone, nitric
44 acid, and acetic acid (Millstein et al. 2004). Similar results were reported for eight areas
45 of Switzerland in which a 10 |ig/m3 average increase in NC>2 exposure was associated
46 with decreases in FVC (Schindler et al. 1998).
15
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1
2 Several recent studies have attempted to describe the correlation between NO2
3 levels and mortality or respiratory symptoms by pooling large datasets from multiple
4 cities or countries. One of these studies used information collected from up to 12 cities in
5 Canada. These authors found that an approximate 20 ppb increase in NO2 was positively
6 associated with a 2.25% increase in mortality (Burnett et al. 2004), intrauterine growth
7 retardation (odds ratio 1.14-1.16) (Liu et al. 2007), a 17.72% increase in SIDS incidence
8 (Dales et al. 2004), increased numbers of hospitalizations due to cardiac disease (Cakmak
9 et al. 2006), and greater asthma hospitalizations in children 6-12 years of age (Lin et al.
10 2003). However, many of the positive findings in Canada were also positively correlated
11 with other pollutants such as particulate matter, ozone, and sulfur dioxide. Similarly, a
12 significant association of NO2 with cardiovascular and respiratory mortality was found in
13 30 European cities (Samoli et al. 2006) and in nine French cities (Le Tertre et al. 2002),
14 but evidence of confounding effects of black smoke, sulfur dioxide, and/or ozone were
15 also found in both studies.
16
17 Asthma and allergy prevalence in conjunction with NO2 levels have also been
18 assessed in multiple city or country studies. Positive correlations were found for asthma
19 attacks, tightness in the chest, wheeze, and/or allergic rhinitis in children from eight
20 Japanese communities (Shima et al. 2002) and in 13 areas of Italy with the most
21 pronounced effects in the warmer Mediterranean areas (de Marco et al. 2002). An
22 increased incidence of morning symptoms was associated with a 6-day average increase
23 in NO2 (odds ratio 1.48) in asthmatic children from eight US cities (Mortimer et al.
24 2002). In a cross-sectional study of five countries, long-term NO2 concentrations were
25 correlated with sensitivity to inhaled allergens, but not to prevalence of bronchitis or
26 asthma (Pattenden, et al. 2006). No association was found between NO2 concentrations
27 and asthma, allergic rhinitis, and atopic dermatitis in children from six French cities
28 (Penard-Morand et al. 2005).
29
30 Epidemiological studies associating indoor NO2 have also been inconclusive.
31 One study found no evidence of any short-term association between prevalence of
32 respiratory symptoms in infants and median indoor and outdoor NO2 levels of 6.8 and
33 12.6 ppb, respectively (Farrow et al. 1997). Similarly, no associations were found with
34 indoor NO2 and wheeze and asthma in children from seven Japanese communities (Shima
35 and Adachi 2000). Other studies found a significant increase in the occurrence of sore
36 throat, colds, and absences from school among children exposed to hourly peak levels of
37 >80 ppb NO2 from unvented gas heating in the classrooms (Pilotto et al. 1997), increased
38 respiratory illness in children from homes using gas cooking where NO2 concentrations
39 in the children's bedroom ranged from 4-169 ppb (Florey et al. 1979), and slight
40 decreases in forced vital capacity and peak expiratory flow among adult asthmatics
41 exposed to >0.3 ppm while cooking on a gas range (Goldstein et al. 1988). Similarly,
42 Neas et al. (1991) found that a 15 ppb increase in household annual NO2 mean was
43 associated with an increased cumulative incidence of attacks of shortness of breath with
44 wheeze, chronic wheeze, chronic cough, chronic phlegm, or bronchitis in children.
45
46 As part of a review of the National Ambient Air Quality Standards (NAAQS) for
16
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 NC>2, U.S. EPA (1995) conducted a meta-analysis of studies which examined the
2 respiratory effects on children living in homes with gas stoves. Conclusions drawn from
3 that analysis were that children ages 5-12 years old had an increased risk of about 20%
4 for developing respiratory symptoms and disease with each increase of 0.015 ppm in
5 estimated 2-week average NC>2 exposure (mean weekly concentrations in bedrooms
6 0.008-0.065 ppm) and that no evidence for increased risk was found for infants <2 years
7 old. Several limitations of this meta-analysis have been noted, including the following:
8 uncertainty between monitored vs. actual exposure concentration; peak and average
9 exposures could not be distinguished by the method used; and confounding effects of
10 other gas combustion by-products. In context of the NAAQS review it was noted that
11 indoor exposures do not mimic outdoor exposures (U.S. EPA 1995).
12
13 Several occupations result in exposure to NC>2 concentrations higher than ambient
14 levels. In diesel bus garage workers, NC>2 concentrations of >0.3 ppm, along with
15 respirable particulates, were associated with work-related symptoms of cough, itching,
16 burning or watering eyes, difficult breathing, chest tightness, and wheeze; but, there were
17 no reductions in pulmonary function (Gamble et al. 1987). In contrast, no relationship
18 was found between respiratory symptoms or decline in FEVi among British coalminers
19 and exposure to peak NC>2 concentrations up to 14 ppm; controls were matched for age,
20 dust exposure, smoking habit, coal rank, and type of work (Robertson et al. 1984). No
21 differences in pulmonary function were noted among shipyard welders exposed to
22 average concentrations of 0.04 ppm oxides of nitrogen (Peters et al. 1973). Slight
23 increases in prevalence of bronchitis (17.2% vs 12.6%) and colds (37.5% vs 30.7%) were
24 noted in traffic officers exposed to automobile exhaust containing mean concentrations of
25 0.045-0.06 ppm NO2 (Speizer and Ferris 1973).
26
27 In conclusion, indoor air quality may be more significant than outdoor air quality
28 to the prevalence of respiratory illness due to NC>2. A review of epidemiology studies
29 which assessed ambient quality (U.S. EPA 1993) yielded insufficient evidence to reach
30 any conclusion about the long- or short-term health effects of NC>2. Further, review of
31 epidemiology studies that assessed indoor air quality in homes with gas stoves, found
32 that meta analysis yielded insufficient evidence that NC>2 had an effect on infants 2 years
33 and younger while several considerations limited the interpretation of the positive
34 results for children aged 5-12 years.
35
36 2.2.3. Experimental Studies
37
38 Healthy Subjects
39
40 The odor threshold for NC>2 in air has been reported as 0.4 ppm for recognition
41 and 4.0 ppm for less than 100% identification (NIOSH 1976). In an experimental
42 study, the odor of NC>2 was perceived by 3/9 volunteers exposed to 0.12 ppm and by
43 8/13 subjects at 0.22 ppm. At concentrations of <4 ppm, the volunteers perceived the
44 odor for 1-10 minutes, but the duration of perception was not directly related to
45 concentration. The olfactory response to NC>2 returned 1-1.5 minutes after cessation of
46 exposure (Henschler et al. 1960). There appears to be a difference between perception
17
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 and recognition level concentrations and the volunteers perceiving the odor at the
2 lowest concentrations were described as "olfactorily sensitive."
3
4 Exposures of healthy individuals to <2 ppm NC>2 have shown no effects on
5 pulmonary function or symptoms. In several studies, healthy men and/or women were
6 exposed to 0.6 ppm NC>2 for 1-3 hours with intermittent or continuous exercise. No
7 significant effects were observed in any study on pulmonary function, cardiovascular
8 function, metabolism, or symptoms of exposure (Folinsbee et al. 1978, Adams et al.
9 1987, Frampton et al. 1991, Hazucha et al. 1994). No changes in pulmonary function
10 occurred following exposure to 1.5 ppm for 3 hours or to a baseline of 0.05 ppm with
11 intermittent peaks of 2 ppm, however, continuous exposure to 1.5 ppm for 3 hours
12 resulted in a slight but significantly greater fall in FEVi and FVC in response to
13 carbachol (Frampton et al. 1991). Pulmonary function was not affected in competitive
14 athletes exposed to 0.18 and 0.30 ppm for 30 minutes during heavy exercise (Kim et al.
15 1991) or in healthy adults exposed to 0.3 ppm for 4 hours with intermittent exercise
16 (Smeglin et al. 1985).
17
18 Studies at higher concentrations of NC>2 indicate an apparent threshold before
19 pulmonary function is affected. No changes in pulmonary function, airway reactivity, or
20 indications of irritation were measured in healthy adults exposed to 1 ppm for 2 hours, 2
21 ppm for 3 hours (Hackney et al. 1978), 2 ppm for 4 hours (Devlin et al. 1992), 3 ppm for
22 2 hours (Goings et al. 1989) or to 2.3 ppm for 5 hours (Rasmussen et al. 1992). Normal
23 subjects exposed to 2 ppm for 1 hour developed an increase in airway reactivity to
24 methacholine challenge without changes in lung volume or pulmonary function
25 (Mohsenin 1988). No statistically significant effects on airway resistance, symptoms,
26 heart rate, skin conductance, or self-reported emotional state were found in healthy
27 volunteers exposed to 4 ppm NO2 for 1 hour and 15 minutes with intermittent light and
28 heavy exercise (Linn and Hackney 1983). However, a significant decrease in mean (n =
29 11) alveolar O2 partial pressure by 8 mm Hg and a significant increase in mean (n = 11)
30 airway resistance from 1.51 to 2.41 cm H2O/(L/s) occurred in healthy volunteers exposed
31 to 5 ppm for 2 hours with 6/11 individuals responding (von Nieding et al. 1979).
32 Similarly, a 10-minute exposure to 4-5 ppm resulted in increased expiratory and
33 inspiratory flow resistance in five healthy males; the effect was greatest 30 minutes after
34 exposure (Abe 1967).
35
36 Henschler et al. (1960) performed several experiments on healthy, male
37 volunteers. They reported that a 2-hour exposure to 20 ppm did not cause any irritation
38 when preceded by several exposures to lower concentrations during the preceding days;
39 however, exposure to 30 ppm for 2 hours caused definite discomfort. Three individuals
40 exposed to 30 ppm for 2 hours perceived an intense odor upon entering the chamber, the
41 odor quickly diminished and was completely absent after 25-40 minutes. One individual
42 experienced a slight tickling of the nose and throat mucous membranes after 30 minutes,
43 the two others after 40 minutes. From 70 minutes on, all subjects experienced a burning
44 sensation and an increasingly severe cough for the next 10-20 minutes, but coughing
45 decreased from 100 minutes on. However, the burning sensation continued and moved
46 into the lower sections of the airways and was finally felt deep in the chest. At this time,
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 marked sputum secretion and dyspnea were noted. Toward the end of the exposure, the
2 subjects reported the exposure conditions to be bothersome and barely tolerable. A
3 sensation of pressure and increased sputum secretion continued for several hours after
4 cessation of exposure (Henschler et al. 1960).
5
6 In a similar experiment (Henschler and Liitke 1963) groups of 4 or 8 healthy,
7 male volunteers were exposed to 10 ppm for 6 hours or to 20 ppm for 2 hours. All
8 subjects upon entering the chamber noted the odor which diminished rapidly. At 20 ppm
9 minor scratchiness of the throat was felt after about 50 minutes and 3/8 experienced slight
10 headaches toward the end of the exposure period. Methemoglobin levels remained
11 within the normal range in all subjects after exposure.
12
13 Biochemical changes in bronchoalveolar lavage fluid (BALF) and blood have also
14 been studied following exposure of healthy adults to NC>2. Exposures to 2 ppm for 4
15 (Devlin et al. 1992) or 6 hours (Frampton et al. 1992) caused an influx of
16 polymorphonuclear leukocytes in BALF, 2.3 ppm for 5 hours resulted in a decrease in
17 serum glutathione peroxidase activity (Rasmusen et al. 1992), 1 and 2 ppm for 3 hours
18 caused a decrease in RBC membrane acetylcholinesterase activity, 2 ppm for 3 hours
19 resulted in an increase in peroxidized RBC lipids and glucose-6-phosphate
20 dehydrogenase activity (Posin et al. 1978), and exposure to 3 or 4 ppm for 3 hours
21 resulted in a decrease in a-1-protease inhibitor activity but not in enzyme concentration in
22 BALF (Mohsenin and Gee 1987). Following exposure to 2 ppm for 4 hours, neutrophilic
23 inflammation was detected in bronchial washings but no changes in inflammatory cells
24 were observed in endobronchial biopsy samples (Blomberg et al. 1997). Mucociliary
25 activity was completely stopped in healthy individuals 45 minutes after a 20-minute
26 exposure to 1.5 and 3.5 ppm (Helleday et al. 1995).
27
28 Asthmatic Subjects
29
30 Studies on the effects of NC>2 on pulmonary function in asthmatics are
31 inconclusive and conflicting. No consistent changes in pulmonary function or reported
32 symptoms have been found in exercising asthmatic adults and adolescents exposed to
33 0.12 or 0.18 ppm for 40 minutes (Koenig et al. 1987), 0.12 ppm for 1 hour at rest (Koenig
34 et al. 1985), 0.2 ppm for 2 hours with intermittent exercise (Kleinman et al. 1983), 0.3
35 ppm for 30 minutes (Rubinstein et al. 1990), 1 hour (Vagaggini et al. 1996), or 4 hours
36 with exercise (Morrow and Utell 1989), 0.5 ppm for 1 hour at rest (Mohsenin 1987), up
37 to 0.6 ppm for 75 minutes with intermittent exercise (Roger et al. 1990), and up to 1 ppm
38 for 4 hours (Sackner et al. 1981). No statistically significant differences between control
39 and NC>2 exposure were found for airway resistance, symptoms, heart rate, skin
40 conductance, or self-reported emotional state for asthmatics exposed to 4 ppm for 75
41 minutes with intermittent exercise (Linn and Hackney 1984).
42
43 Kerr et al. (1978, 1979) studied the effects of NC>2 on pulmonary function and
44 reported other symptoms which were not included in many other studies. The subjects
45 were asked to keep note of symptoms they experienced during exposure to 0.5 ppm for 2
46 hours, specifically cough, sputum, irritation of mucus membranes, and chest discomfort.
19
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 At this concentration the odor of NO2 was perceptible but the subjects became unaware
2 of it after about 15 minutes. Seven of 13 asthmatics reported symptoms with exposure,
3 compared with only one of 10 normal subjects and one of 7 subjects with chronic
4 bronchitis. In the group of asthmatics, two had slight burning of the eyes, one had a
5 slight headache, three reported chest tightness, and one had labored breathing with
6 exercise, compared with slight nasal discharge in the normal and chronic bronchitis
7 individuals. No changes in any pulmonary function tests were found immediately
8 following the exposure.
9
10 Significant group mean reductions in FEVi (-17.3% with NO2 vs -10.0% with air)
11 and specific airway conductance (-13.5% with NO2 vs -8.5% with air) occurred in
12 asthmatics after exercise during exposure to 0.3 ppm for 4 hours and 1/6 individuals
13 experienced chest tightness and wheezing (Bauer et al. 1985). The onset of effects was
14 delayed when exposures were by oral-nasal inhalation compared to oral inhalation; the
15 delay may have resulted from scrubbing within the upper airway. In a similar study, 15
16 asthmatics exposed to 0.3 ppm for 20 minutes at rest followed by 10 minutes of exercise
17 had significantly greater reductions in FEVi (-10% vs -4% with air) and partial expiratory
18 flow rates at 60% of total lung capacity, but no symptoms were reported (Bauer et al.
19 1986). In a preliminary study with 13 asthmatics exposed to 0.3 ppm for 110 minutes,
20 slight cough and dry mouth and throat and significantly greater reduction (-11% vs -7%)
21 in FEVi occurred after exercise, however, in a larger study, no changes in pulmonary
22 function were measured and no symptoms were reported following exposure of 21
23 asthmatics to concentrations up to 0.6 ppm for 75 minutes (Roger et al. 1990). The mean
24 drop in FEVi for asthmatics during a 3-hour exposure to 1 ppm NO2 (-2.5%) with
25 intermittent exercise was significantly greater than the drop during air (-1.3%) exposure;
26 in BALF, levels of 6-keto-prostaglandinia were decreased and levels of thromboxane B2
27 and prostaglandin D2 were increased after NO2 exposure (Torres et al. 1995).
28
29 Studies on the effects of NO2 on airway hyperreactivity in asthmatics have also
30 been inconclusive. Methacholine responsiveness in asthmatics was not increased
31 following exposure to 0.25 ppm for 20 minutes at rest plus 10 minutes of exercise
32 (Torres and Magnussen 1991) or by exposure to 0.1 ppm for 1 hour at rest (Hazucha et
33 al. 1983). Exposure to 0.1 ppm for 1 hour caused an increase of specific airway
34 resistance in 3/20 asthmatics (the remaining 17 individuals had little or no response to
35 NO2 exposure) and enhanced the bronchoconstrictor effect of carbachol in 13/20
36 asthmatics, but the remaining 7 subjects were unaffected. When the study was repeated
37 in 4 individuals (two responders and two non-responders) at 0.2 ppm, the results were
38 variable; the two non-responders were still unaffected, while one responder had an
39 equal response and the other had a greater response to carbachol challenge compared
40 with their responses to 0.1 ppm (Orehek et al. 1976). Slight but significant potentiation
41 of airway reactivity in asthmatics occurred from exposure to 0.5 ppm NO2 for 1 hour
42 followed by methacholine challenge (Mohsenin 1987), 0.3 ppm for 40 minutes
43 followed by isocapnic cold air hyperventilation (Bauer et al. 1986), 0.2 ppm for 2 hours
44 followed by methacholine challenge (Kleinman et al. 1983), and 0.25 ppm for 30
45 minutes followed by isocapnic hyperventilation (Torres and Magnussen 1990). A
46 significantly greater fall in FEVi from challenge with house dust mite antigen was
20
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 reported for asthmatics compared to controls (-7.76% vs -2.85%) following exposure to
2 0.4 ppm for 1 hour (Tunnicliffe et al. 1994), but no significant changes were found in a
3 similar study using a 6-hour exposure (Devalia et al. 1994). Exposure of asthmatics to
4 0.4 ppm for 3 hours significantly decreased the amount of inhaled allergen required to
5 decrease FEVi by 20% but no changes in airway responsiveness occurred following
6 exposure to 0.2 ppm for 6 hours; these results suggest a concentration threshold rather
7 than a duration effect (Jenkins et al. 1999).
8
9 Folinsbee (1992) conducted a meta-analysis of twenty studies which measured
10 airway responsiveness in asthmatics following NO2 exposure. Eight different agents
11 were used in these studies to induce non-specific airway responsiveness and the analysis
12 was limited to exposures in the range of 0.2 to 0.3 ppm. The fraction of asthmatic
13 subjects with an increase in airway responsiveness was significant (p < 0.01) following
14 exposures at rest, but not with exercise. When only those studies which used a
15 cholinergic agonist were analyzed, similar results were found in that a greater proportion
16 of subjects showed an increased response in resting exposures than in exercising
17 exposures.
18
19 Subjects with Chronic Lung Disease
20
21 The results of studies on the effects of NO2 on pulmonary function in patients
22 with chronic lung disease or bronchitis are also conflicting. No significant differences in
23 pulmonary function or symptom reporting were observed in patients with chronic
24 respiratory illness exposed to 0.3 ppm for 4 hours at rest (Hackney et al. 1992), in
25 patients with chronic obstructive pulmonary disease (COPD) exposed to NO2 levels up to
26 2 ppm for 1 hour with intermittent exercise (Linn et al. 1985), and in patients with
27 chronic bronchitis exposed to 0.5 ppm for 2 hours with exercise (Kerr et al. 1978, 1979).
28 In contrast to these reports, forced expiratory volume of COPD patients significantly
29 decreased from 18.8 L after air to 13.6 L after exposure to 0.3 ppm for 1 hour (Vagaggini
30 et al. 1996). A significant reduction in forced vital capacity that progressed during
31 exercise (-1.2 to -8.2%) occurred in elderly COPD patients exposed to 0.3 ppm for 4
32 hours while no effects were seen in an age and gender matched healthy control group
33 (Morrow and Utell 1989, Morrow et al. 1992).
34
35 The effects of NO2 on respiratory gas exchange were investigated in patients with
36 chronic bronchitis. Inhalation of 4 and 5 ppm for 15-60 minutes significantly decreased
37 the CO diffusing capacity and arterial pO2 with no progressive changes noted over time.
38 Exposure to 5 ppm over 15 minutes resulted in an average decrease in CO diffusion
39 capacity of 3.8 mL/min/torr and a decrease in arterial pO2 from an average of 76.5 to 71.3
40 torr. A slight, but statistically significant, increase in airway resistance (approximately
41 20-30% above the initial value) was measured at concentrations of 1.6-5 ppm for 5
42 minutes; no effects occurred at or below 1.5 ppm (von Nieding et al. 1973, von Nieding
43 and Wagner 1979).
44
45 2.3. Developmental/Reproductive Toxicity
46
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 No information was found regarding the developmental or reproductive toxicity
2 of NC>2 in humans.
3
4 2.4. Genotoxicity
5
6 No information was found regarding the genotoxicity toxicity of NC>2 in humans.
7
8 2.5. Carcinogenicity
9
10 No information was found regarding the carcinogenicity of NC>2 in humans.
11
12 2.6. Summary
13
14 In humans, exposure to >15 ppm NC>2 in healthy individuals causes immediate
15 irritation with pulmonary edema followed by a latent period of apparent recovery. A
16 second phase of symptoms can occur after several hours or several days with the
17 development of fever with progressively more severe dyspnea, cyanosis, and cough, and
18 inspiratory and expiratory rales. An estimation of the concentration causing death in
19 humans is approximately >150 ppm, but no duration of exposure was given. Most case
20 reports do not contain concentrations or durations of exposure, however, welders exposed
21 to 30 and 90 ppm for 40 minutes experienced varying degrees of dyspnea, cough,
22 headache, chest tightness, nausea, and cyanosis with hospitalization required for
23 pulmonary edema, confirmed by X-ray, at the higher concentration (Norwood et al. 1966,
24 Morley and Silk 1970). Similar symptoms and findings of respiratory complaints were
25 reported following release of a cloud of ^64 from a railroad tank car (Bauer et al. 1996).
26
27 Epidemiological studies on the long-term effects of elevated NC>2 are
28 conflicting. It is likely that increases in respiratory illnesses are due to NC>2 in
29 combination with other pollutants and that short-term peak concentrations are more
30 detrimental than chronic, low-level exposures. Evidence suggests that children ages 5-
31 12 have a greater risk for developing respiratory disease from long-term exposure to
32 higher concentrations, but that infants do not.
33
34 Experimental studies with both healthy and asthmatic individuals are
35 inconclusive. Negative results have been obtained in many studies with exposures up
36 to 4 ppm for 1 hour, however, other studies report positive effects on pulmonary
37 function at lower concentrations. It should be noted that in the studies which found
38 statistically significant differences with NC>2 exposure, the changes were within 10% of
39 the measured value after air only exposure and of questionable biological significance
40 even for asthmatics. However, the available evidence also suggests that asthmatics
41 may experience an increase in airway responsiveness from concentrations of 0.2-0.3
42 ppm.
43
44 3. ANIMAL TOXICITY DATA
45 3.1. Acute Lethality
46
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 Acute lethality data from NO2 exposures were located for several species. One
2 group of investigators (Hine et al. 1970) studied the effects of varying concentration and
3 duration of exposure in five different species of laboratory animal; these results are
4 described separately by species below and summarized in Table 4 at the end of this
5 section. In this study, deaths generally occurred within 2-8 hours after exposure and the
6 majority within 24 hours. Additional data from rabbit, rat, and mouse studies were
7 available and are in good agreement with the results of the Hine et al. study. LCso values
8 for N2O4 were listed for four species, but duration of exposure was not given; effects
9 were similar to those described following NO2 exposure.
10
11 3.1.1. Dogs
12
13 Greenbaum et al. (1967) exposed mongrel dogs (n = I/exposure) to 0.1% (1000
14 ppm) NO2 for 136 minutes, 0.5% (5000 ppm) for 5-45 minutes, or to 2% (20,000 ppm)
15 for 15 minutes. All dogs that were exposed to either 0.5% for 35-45 minutes or to 2% for
16 15 minutes died. Respiration became shallow and gasping with death due to pulmonary
17 edema. Fluid was visible in the tracheobronchial tree at necropsy. Cyanosis due to
18 methemoglobin formation (78%) was noted in one animal given 2% for 15 minutes. At
19 concentrations of 0.5% and 2%, arterial pO2 and systemic arterial pressure were reduced.
20 The authors stated that pulmonary edema was caused by the action of nitrogen dioxide on
21 the alveolar lining fluid, forming nitric and nitrous acids which, in turn, cause denaturing
22 of proteins, rupture of lysosomes, and the development of chemical pneumonitis.
23
24 Hine et al. (1970) studied the effects of varying concentration and duration of
25 NO2 exposure on mongrel dogs. Animals (n = 1-4) were exposed to 5-250 ppm NO2 for
26 periods of 30 minutes to 24 hours. At concentrations of >40 ppm, signs of toxicity
27 included lacrimation, reddening of the conjunctivae, and increased respiration which
28 became labored and difficult as the concentration increased. Mortalities were first
29 observed at 75 ppm for 4 hours (Table 4). Terminally, respiration became gasping and
30 spasmodic and lung edema was observed at necropsy. Histological findings in the lungs
31 of decedents included bronchiolitis, desquamated bronchial epithelium, infiltration by
32 polymorphonuclear cells, and edema.
33
34 Kushneva and Gorshkova (1999) list an LC50 of 260 mg/m3 (70 ppm) for N2O4,
35 with the cause of death pulmonary edema; duration of exposure was not given and no
36 experimental details were included.
37
38 3.1.2. Rabbits
39
40 The 15-minute LCso for the rabbit (strain not specified; n = 5) was 315 ppm.
41 Clinical signs of toxicity included severe respiratory distress, eye irritation, 10-15% body
42 weight suppression for two days, and death; time to death varied from 30 minutes to 3
43 days. Gross pathology revealed darkened areas on the surface of the lungs.
44 Histopathology of the lungs of the survivors 7 and 21 days after exposure showed focal
45 accumulation of intraalveolar macrophages, some proliferation of the alveolar lining
46 epithelium, and varying amounts of inflammatory cells (Carson et al. 1962).
23
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1
2 Hine et al. (1970) studied the effects of varying concentration and duration of
3 NO2 exposure on rabbits (strain not specified). Animals (n = 2-8) were exposed to 5-200
4 ppm NO2 for 30 minutes up to 24 hours. At concentrations of >40 ppm, signs of toxicity
5 included lacrimation, reddening of the conjunctivae, and increased respiration which
6 became labored and difficult as the intoxication increased. One mortality was observed
7 at 75 ppm for 60 minutes but none occurred at 2 hours making the death after the 1 hour
8 exposure questionable (Table 4). Terminally, respiration became gasping and spasmodic
9 and lung edema was observed at necropsy. Histological findings in the lungs of
10 decedents included bronchiolitis, desquamated bronchial epithelium, infiltration by
11 polymorphonuclear cells, and edema.
12
13 In a similar study, rabbits (strain not specified; n = 3) were exposed to 125, 175,
14 250, 400, 600, or 800 ppm NO2 for 10 minutes (Meulenbelt et al. 1994). Two of three
15 animals given 800 ppm died 7-21 hours after exposure. Lung weights were significantly
16 higher and lung homogenates contained greater amounts of protein and higher levels of
17 lactate dehydrogenase (LDH), glutathione peroxidase, and glucose-6-phosphate
18 dehydrogenase activity in animals exposed to >250 ppm. Bronchoalveolar lavage fluid
19 from animals exposed to > 175 ppm contained greater amounts of protein and albumin,
20 and higher levels of LDH and angiotensin converting enzyme activity than unexposed
21 controls and all treated groups had increased numbers of neutrophilic leucocytes. Dose-
22 related increases in severity of centriacinar catarrhal pneumonitis, macrophage influx,
23 and neutrophilic leucocytes were observed on histopathological examination of the lungs.
24 Edema occurred at >250 ppm, subpleural hemorrhaging at >400 ppm, and desquamation
25 of the bronchiolar epithelium was seen at >600 ppm.
26
27 Kushneva and Gorshkova (1999) list an LC50 of 320 mg/m3 (86 ppm) for N2O4,
28 with the cause of death pulmonary edema; duration of exposure was not given and no
29 experimental details were included.
30
31 3.1.3. Guinea Pigs
32
33 Hine et al. (1970) also studied the effects of varying concentration and duration of
34 exposure in the guinea pig (strain not specified). Animals (n = 2-6) were exposed to 5-
35 200 ppm NO2 for 30 minutes up to 8 hours. At concentrations of >40 ppm, signs of
36 toxicity included lacrimation, reddening of the conjunctivae, and increased respiration
37 which became labored and difficult as the intoxication increased. Mortalities were first
38 observed at 50 ppm for 1 hour (Table 4). Terminally, respiration became gasping and
39 spasmodic and lung edema was observed at necropsy. Histological findings in the lungs
40 of decedents included bronchiolitis, desquamated bronchial epithelium, infiltration by
41 polymorphonuclear cells, and edema.
42
43 To determine the sensitivity of adult and neonate animals to NO2 inhalation,
44 Duncan-Hartley guinea pigs aged 5, 10, 21, 45, 55, and 60 days were exposed
45 continuously for 3 days to 2 or 10 ppm (Azoulay-Dupuis et al. 1983). A total of 17-27
46 animals were studied in each age group and litter exposures prior to weaning included the
24
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 dam. At 10 ppm, clinical signs of toxicity in adults over 45 days old included moving
2 with difficulty, reduced food and water consumption, and hyperventilation. Body weight
3 gain was decreased until 21 days and body weight was reduced after 45 days in all
4 exposed animals. These effects were most pronounced in the dams. Mortality in the
5 high-exposure group increased with age with 4% of 5-day olds, up to 60% of 55-day
6 olds, and 67% of dams dying. Further, most of the older animals died after the first 24
7 hours of exposure whereas the younger animals died later in the 3-day period. At 2 ppm,
8 lung histopathology was normal until animals were 45 days of age when thickening of the
9 alveolar walls, infiltration by PMNs, and alveolar edema were observed; in dams
10 bronchioles were devoid of cilia in some areas. At 10 ppm, guinea pigs of all ages were
11 affected by these changes which were more pronounced in older animals.
12
13 3.1.4. Rats
14
15 Five-, 15-, 30-, and 60-minute LCso values for the male rat (100-120 g; strain
16 not specified; n = 10) are 416, 201, 162, and 115 ppm, respectively. Clinical signs of
17 toxicity included severe respiratory distress, eye irritation, 10-15% body weight
18 suppression, and death; time to death varied from 30 minutes to 3 days. Gross
19 pathology revealed darkened areas on the surface of the lungs, and, in some instances,
20 purulent nodules involving the entire lungs of some of the survivors (Carson et al.
21 1962).
22
23 An older study (Gray et al. 1954) reported LCso values for male rats (200-300 g;
24 strain not specified; n = 10) of 1445 ppm for 2 minutes, 833 ppm for 5 minutes, 420
25 ppm for 15 minutes, 174 ppm for 30 minutes, 168 ppm for 60 minutes, and 88 ppm for
26 240 minutes. Deaths were attributed to pulmonary edema. The differences in LCso
27 values between this study and Carson et al. (1962) may be due to the differences in size
28 and age of the rats used in each.
29
30 Meulenbelt et al. (1992a,b) investigated the effects of both concentration and
31 duration of exposure in Wistar rats. The effect of concentration was studied by exposing
32 6-9 rats/group to 25, 75, 125, 175 or 200 ppm NC>2 for 10 minutes. No signs of toxicity
33 were observed at 25 ppm. Stertorous respiration was heard in animals exposed to 175
34 and 200 ppm. Rats exposed to >75 ppm had significantly increased lung weight, and
35 subpleural hemorrhages and pale discol orations of the lung were observed grossly.
36 Histologically, the lungs from these animals showed atypical pneumonia, edema, focal
37 desquamation of the terminal bronchiolar epithelium, increased numbers of macrophages
38 and neutrophilic leucocytes, and interstitial thickening of the centriacinar septa (175 and
39 200 ppm only) with the severity increasing at the higher concentrations. One rat died 14-
40 20 hours after exposure in both the 175 ppm and 200 ppm groups. Biochemical changes
41 in bronchoalveolar lavage fluid included concentration-dependent increases in protein
42 and albumin concentrations, angiotensin converting enzyme activity, p-glucuronidase
43 activity, and neutrophilic leukocytes.
44
45 Duration of exposure was investigated by exposing 6 rats/group to either 175 ppm
46 for 10, 20, or 30 minutes or to 400 ppm for 5, 10, or 20 minutes (Meulenbelt et al.
25
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 1992a,b). Stertorous respiration was heard in animals for all exposure times at both
2 concentrations and lung weight was significantly higher than that of the controls at all
3 exposure durations. At 175 ppm, 5/6 rats died in the 20 and 30 minute groups and at 400
4 ppm, 6/6 rats died in the 10 and 20 minute groups. Necropsy revealed foamy,
5 seroanguinous fluid in the trachea, subpleural bleeding, and pale discoloration.
6 Histological alterations were similar to those described above. Methemoglobin levels,
7 measured after exposure to 175 ppm for 10 minutes, were not elevated, but plasma nitrate
8 levels were significantly higher than controls.
9
10 Hine et al. (1970) also studied the effects of varying concentration and duration of
11 exposure in Long-Evans rats. Animals (n= 4-31) were exposed to 5-250 ppm NO2 for
12 durations of 30 minutes up to 24 hours. At concentrations of >40 ppm, signs of toxicity
13 included lacrimation, reddening of the conjunctivae, and increased respiration which
14 became labored and difficult as the intoxication increased. Mortalities were first
15 observed at 50 ppm for 24 hours (Table 4). Terminally, respiration became gasping and
16 spasmodic and lung edema was observed at necropsy. Histological findings in the lungs
17 of decedents included bronchiolitis, desquamated bronchial epithelium, infiltration by
18 polymorphonuclear cells, and edema.
19
20 Kushneva and Gorshkova (1999) list an LC50 of 105 mg/m3 (28 ppm) for N2O4,
21 with the cause of death pulmonary edema; duration of exposure was not given and no
22 experimental details were included.
23
24 3.1.5. Mice
25
26 BALB/c mice (n = 5-7) were exposed to 5, 20, or 40 ppm NO2 for 12 hours
27 (Hidekazu and Fujio 1981). Body weight was markedly decreased 1 and 2 days after
28 exposure to 20 and 40 ppm and 3/38 (7.8%) animals exposed to 40 ppm died within 2
29 days of the exposure.
30
31 Hine et al. (1970) studied the effects of varying concentration and duration of
32 exposure in Swiss-Webster mice. Animals (n= 5-14) were exposed to 5-250 ppm NO2
33 for durations of 30 minutes up to 24 hours. At concentrations of >40 ppm, signs of
34 toxicity included lacrimation, reddening of the conjunctivae, and increased respiration
35 which became labored and difficult as the intoxication increased. Mortalities were first
36 observed at 50 ppm for 24 hours (Table 4). Terminally, respiration became gasping and
37 spasmodic and lung edema was observed at necropsy. Histological findings in the lungs
38 of decedents included bronchiolitis, desquamated bronchial epithelium, infiltration by
39 polymorphonuclear cells, and edema.
40
41 Kushneva and Gorshkova (1999) list an LC50 of 190 mg/m3 (51 ppm) for N2O4,
42 with the cause of death pulmonary edema; duration of exposure was not given and no
43 experimental details were included.
26
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NITROGEN OXIDES
NAC/Interim (NO2)/Proposed (N2O4): 12/2008
TABLE 4: Summary of NO2 Mortality for Five Species
Cone.
(ppm)
50
75
100
150
200
Time (hr)
1
6
24
1
2
4
8
0.5
2
4
8
0.5
1
2
4
0.08
0.17
0.33
0.50
Rat
0/17
0/12
3/10
3/31
1/12
7/12
12/12
0/5
8/8
29/29
2/10
10/13
10/12
4/4
6/12
8/12
5/5
4/4
Mouse
0/5
0/5
5/10
1/6
2/6
5/6
6/6
2/10
13/14
10/10
10/10
-
4/6
6/6
6/6
Guinea Pig
1/6
4/6
1/4
3/4
2/4
4/4
1/2
3/4
3/4
3/3
2/2
Rabbit
0/4
0/4
1/8
0/6
2/8
6/8
1/3
2/4
3/4
1/6
3/4
0/2
1/2
2/4
Dog
0/1
0/2
0/2
0/2
1/3
1/4
0/2
1/3
2/2
2/3
2/2
2
3
4
5
6
7
Data from Hine et al. 1970, p.
majority within 24 hours.
3
3
.2.
.2.
Nonlethal Toxicity
1. Monkeys
206; deaths generally occurred within 2-8 hours after exposure and the
9 Squirrel monkeys (n = 2-6/group) were exposed to 10-50 ppm NC>2 for 2 hours
10 with respiratory function monitored during exposure (Henry et al. 1969). Exposure to 35
11 or 50 ppm resulted in a markedly increased respiratory rate and decreased tidal volume
12 during exposure which returned to normal by the seventh day post exposure. Only slight
13 effects on respiratory function were noted at 15 and 10 ppm. Mild histopathological
14 changes in the lungs were noted after exposure to 10 and 15 ppm, however, marked
15 changes in lung structure were observed after exposure to 35 and 50 ppm. At 35 ppm,
16 areas of the lung were collapsed with basophilic alveolar septa, in other areas the alveoli
17 were expanded with septal wall thinning, and the bronchi were moderately inflamed with
18 some proliferation of the surface epithelium. At 50 ppm, extreme vesicular dilatation of
19 alveoli or total collapse was observed, lymphocyte infiltration was seen with extensive
20 edema, and surface erosion of the epithelium of the bronchi was observed. In addition to
21 the effects on the lungs, interstitial fibrosis (35 ppm) and edema (50 ppm) of cardiac
22 tissue, glomerular tuft swelling in the kidney (35 and 50 ppm), lymphocyte infiltration in
23 the kidney and liver (50 ppm), and congestion and centrilobular necrosis in the liver (50
24 ppm) were observed. Although no animals died following the single exposure to 50 ppm,
25 one animal died following a second exposure two months after the first exposure
26 suggesting that some of the lesions were irreversible.
27
28 3.2.2. Dogs
27
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1
2 Carson et al. (1962) conducted a series of experiments on dogs (strain not
3 specified; n = 2) at target concentrations of NO2 approximately 50% and 25% of the
4 LC50 for the rat (see Sec. 3.1.5). The actual analyzed concentrations varied slightly, but
5 were within 10% of target. Dogs exposed to 164 ppm for 5 minutes, 85 ppm for 15
6 minutes, or 53 ppm for 60 minutes (approximately 50% of the rat LCso) had some
7 respiratory distress during exposure, a mild cough, and eye irritation all of which cleared
8 within two days after exposure. Dogs exposed to 125 ppm for 5 minutes, 52 ppm for 15
9 minutes, or 39 ppm for 60 minutes (approximately 25% of the rat LCso) showed only
10 mild sensory effects. No gross or microscopic lesions were noted in any dog.
11
12 Greenbaum et al. (1967) exposed mongrel dogs (n = 1) to 0.1% (1000 ppm) NO2
13 for 136 minutes or to 0.5% (5000 ppm) for 5-45 minutes. A concentration of 0.1% did
14 not cause death and the one dog exposed to this concentration remained in good
15 condition throughout the exposure. Exposures to 0.5% for 15 and 22 minutes were not
16 lethal but resulted in respiratory distress which gave rise to anxiety for about 2 hours
17 then resolved without therapy. Histopathologic examination of the lungs was not
18 performed.
19
20 No treatment-related changes in behavior or clinical signs were observed in
21 mongrel dogs (n = 1) exposed to 10-40 ppm NO2 for 6 hours (Henschler and Liitke
22 1963).
23
24 Mongrel dogs (number of animals not stated) exposed to 20 ppm NO2 for up to 24
25 hours showed minimal signs of irritation and changes in behavior. Microscopic lesions
26 were described as questionable evidence of lung congestion and interstitial inflammation
27 for up to 48 hours postexposure (Hine et al. 1970).
28
29 Pulmonary ultrastructural changes were examined in beagle dogs (n = 1) exposed
30 to 3-16 ppm NO2 for 1 hour (Dowell et al. 1971). Intraalveolar edema occurred in most
31 dogs exposed to >7 ppm and was associated with impaired surfactant activity and lung
32 compliance. Ultrastructural alterations included wide-spread bleb formation, loss of
33 pinocytic vesicles, and mitochondrial swelling of endothelial cells. Exposure to 3 ppm
34 resulted in bleb formation in the alveolar endothelium (observed by EM) without
35 biochemical or physiological changes.
36
37 3.2.3. Rabbits
38
39 Rabbits (strain not specified; number of animals not given) exposed to 20 ppm
40 NO2 for up to 24 hours showed minimal signs of irritation and changes in behavior.
41 Microscopic lesions were described as questionable evidence of lung congestion and
42 interstitial inflammation for up to 48 hours postexposure (Hine et al. 1970).
43
44 Rabbits exposed to 10 ppm NO2 for 2 hours showed accelerated alveolar
45 particle clearance (Vollmuth et al. 1986) and altered pulmonary arachidonic acid
46 metabolism (Schlesinger et al. 1990). Continuous exposure of rabbits to 3.6 ppm
28
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 NC>2 for 6 days did not cause morphological changes in the lungs (Hugod 1979).
2
3 Rabbits (strain, sex, number not given) exposed continuously for up to 20
4 hours to 7, 14, or 28 ppm NO2 had an increase in polymorphonuclear leukocytes in
5 the lavage fluid throughout the exposure (Gardner et al. 1977).
6
7 3.2.4. Sheep
8
9 Lung mechanics, hemodynamics, and blood chemistry were assessed in crossbred
10 sheep (n = 5-6) exposed by nose-only or lung-only (to mimic mouth breathing) to 500
11 ppm NC>2 for 15 minutes; in another group exposed by lung-only, bronchoalveolar lavage
12 fluid content was examined after a 20 minute exposure to 500 ppm (Januszkiewicz and
13 Mayorga 1994). No changes in hemodynamics or blood chemistry occurred in either
14 group. Mean inspired minute ventilation was significantly increased, resulting in
15 increased breathing rate and decreased mean tidal volume, in the lung-only exposure
16 group, but not the nose-only group. Both nose-only and lung-only exposure groups had
17 significantly increased lung resistance and decreased dynamic lung compliance.
18 Histopathologic examination of tissue from the lung-only exposed group revealed
19 exudative fluid distributed in a patchy lobular pattern with mild neutrophil infiltration;
20 little evidence of exudation was seen in the nose-only exposed group. Epithelial cell
21 number and total protein in bronchoalveolar lavage fluid were significantly increased in
22 the NC>2 exposed animals while macrophage number was decreased.
23
24 Airway reactivity to aerosolized carbachol was evaluated in crossbred sheep (n =
25 4-10) exposed to 7.5 or 15 ppm NC>2 for 2 hours (Abraham et al. 1980). Group means for
26 pulmonary resistance, bronchial reactivity to carbachol, and static lung compliance were
27 similar to those from controls at both concentrations. However, following exposure to
28 7.5 ppm NC>2, 5 of 9 animals showed an increase in pulmonary resistance after carbachol
29 exposure of 57% above baseline and following exposure to 15 ppm NC>2, 9 of 10 animals
30 responded with either bronchoconstriction or hyperreactivity. In a concurrent
31 experiment, sheep were exposed to 15 ppm NC>2 for 4 hours (Abraham et al. 1980).
32 Mean pulmonary resistance was significantly increased from the preexposure value, but
33 there were no changes in pulmonary hemodynamics or clinical signs of distress.
34
35 3.2.5. Guinea Pigs
36
37 Guinea pigs (strain not specified; number of animals not given) exposed to 20
38 ppm NC>2 for up to 24 hours showed minimal signs of irritation and changes in behavior.
39 Microscopic lesions were described as questionable evidence of lung congestion and
40 interstitial inflammation for up to 48 hours postexposure (Hine et al. 1970). Guinea pigs
41 exposed to 9 and 13 ppm for 2 hours or to 5.2 and 6.5 ppm for 4 hours had significantly
42 increased respiratory rate and decreased tidal volume with complete recovery after
43 cessation of exposure (Murphy et al. 1964).
44
45 Hartley guinea pigs (n = 5-16) maintained on an ascorbic acid-deficient diet had
46 increased lung lavage fluid protein following exposure to 4.8 ppm NC>2 for 3 hours and
29
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 increased wet lung weight, increased nonprotein sulfhydryl and ascorbic acid content of
2 the lungs, and decreased a-tocopherol content of the lungs following exposure to 4.5 ppm
3 for 16 hours. These changes were not seen in animals maintained on normal guinea pig
4 diets (Hatch et al. 1986). Similarly, vitamin C-deficient male Hartley guinea pigs (n = 3-
5 12) exposed to 1, 3, or 5 ppm for 72 hours had significantly increased protein and lipid
6 content in lavage fluid (Selegrade et al. 1981). No effects were seen at 0.4 ppm. At 5
7 ppm, 50% of the animals died and histopathology revealed multifocal interstitial
8 pneumonia. When the exposure to 5 ppm was shortened to 3 hours, lavage protein was
9 increased with the peak effect 15 hours post-exposure.
10
11 Guinea pigs (strain not specified; n = 12-18) were exposed to 20, 40, or 70 ppm
12 NO2 for 30 minutes followed by a 30-minute exposure to aerosolized albumin; this
13 regimen was repeated 5-7 times at intervals of several days (Matsumura 1970). During
14 the first exposure to 70 ppm, labored breathing, though not severe, was observed in
15 "some" animals, but was not seen with subsequent exposures. Immediately after the fifth
16 exposure to antigen, one-half of the animals in the 70 ppm group showed enhanced
17 airway sensitization (anaphylactic attacks). No effects were seen at 20 or 40 ppm.
18
19 Changes in airway responsiveness to histamine were investigated in Hartley
20 guinea pigs (number of animals not given) exposed to 7-146 ppm NO2 for 1 hour
21 (Silbaugh et al. 1981). Pulmonary function measurements and histamine challenge tests
22 were performed 2 hours before and at about 10 minutes, 2 and 19 hours after NO2
23 exposure. At 10 minutes after exposure, increased sensitivity to histamine occurred at
24 concentrations >40 ppm but returned to baseline thereafter. Concentration-related
25 significantly increased breathing frequency and decreased tidal volume were measured
26 at 10 minutes (exact concentrations not specified) and remained correlated with
27 concentration at 2 and 19 hours.
28
29 3.2.6. Hamsters
30
31 Syrian golden hamsters (n = 5) were administered 28 ppm NO2 for 6, 24, or 48
32 hours and histopathological changes in the lungs were examined by light and electron
33 microscopy (Case et al. 1982, Gordon et al. 1983). The bronchiolar epithelium showed
34 ciliary loss and surface membrane damage, loss of ciliated cells, and epithelial flattening
35 at 24 and 48 hours and epithelial hyperplasia, nonciliated cell hypertrophy, and loss of
36 tight junctions between type I pneumocytes at 48 hours.
37
38 3.2.7. Ferrets
39
40 Weanling domestic ferrets (n = 4-6), 6 weeks of age, were exposed to 5, 10, 15,
41 or 20 ppm NO2 for 4 hours (Rasmussen 1992). A transient inflammatory response was
42 evident as a significantly increased number of neutrophils in the lavage fluid up to 48-
43 hours postexposure at all concentrations. Morphometrically, dose-related decreased
44 alveolar size and thickened alveolar walls indicative of exposure were observed in the
45 lungs.
46
30
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 3.2.8. Rats
2
3 Only one study was found in which rats were exposed to N2O4 and pulmonary
4 lesions were similar to those described following NO2 exposure. Male Wistar rats
5 exposed to 43 ppm N2O4 for 15 minutes had increased lung weight, lung edema, and
6 hemorrhaging (Yue et al. 2004). The chamber atmosphere was generated by injecting
7 liquid N2O4 and heating to evaporate it. Thus it is likely that much of the dimer was
8 converted to NO2.
9
10 Pulmonary injury from NO2 as indicated by increases in lung weight was assessed
11 in male Fischer 344 rats (n = 6-12) following exposure to 10, 25, or 50 ppm for 5, 15, or
12 30 minutes or to 100 ppm for 5 or 15 minutes (Stavert and Lehnert 1990). No significant
13 changes in lung weight occurred in rats exposed to 10 ppm for 30 minutes or to 25-50
14 ppm for up to 15 minutes. Significant increases in lung wet weight and right cranial lobe
15 dry weight were found following exposure to 50 ppm for 30 minutes or to 100 ppm for 5
16 and 15 minutes. However, histological evidence of lung injury was seen in animals
17 exposed to 25 ppm for 30 minutes, 50 ppm for >5 minutes, and 100 ppm for 5 and 15
18 minutes. Findings included accumulation of fibrin, increased numbers of
19 polymorphonuclear leukocytes and macrophages, extravasated erythrocytes, and type II
20 pneumocyte hyperplasia, the severity of which increased with concentration and duration
21 of exposure.
22
23 In a more expanded study, Lehnert et al. (1994) determined that exposure
24 concentration was more important than exposure duration in the severity of lung injury.
25 Male Fischer 344 rats (n = 8-12) were exposed to 25, 50, 75, 100, 150, 200, or 250 ppm
26 NO2 for durations ranging from 5-30 minutes. Lung wet weight was significantly
27 increased following exposure to > 150 ppm for 5 minutes, 100 ppm for 15 minutes, or 75
28 ppm for 30 minutes and further increases were observed as exposure duration increased.
29 The pulmonary edematous response to a given concentration was not proportional to
30 duration, however, increasing concentrations produced proportional increases in lung wet
31 weight when similar exposure durations were compared. Histologically, fibrin and type II
32 cell hyperplasia were observed following 5-minute exposures to >50 ppm the severity
33 increased proportionally to concentration. As further confirmation of concentration-
34 dependent lung injury, rats were exposed to 1-minute bursts of 500-2000 ppm. The
35 severity of the resulting pulmonary edema (as measured by lung wet weight) was directly
36 proportional to exposure concentration. The authors concluded that brief exposures to
37 high concentrations of NO2 are more injurious than longer duration exposures to lower
38 concentrations. Dietary taurine (an antioxidant) was not protective against the increase in
39 lung wet weight and exercise potentiated the severity of the pulmonary edema.
40
41 The concentration-dependent response of the lung to NO2 was confirmed in
42 another study in which Sprague-Dawley rats (n = 5-6) were exposed to 3.6-14.4 ppm for
43 durations of 6-24 hours/day for 3 days (Gelzleichter et al. 1992). Increases in protein
44 content and cell types in lavage fluid demonstrated that the magnitude of lung injury was
45 a function of exposure concentration.
46
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 Carson et al. (1962) conducted a series of experiments at NO2 concentrations
2 approximating 50%, 25%, and 15% of the rat LC50 levels. At the 50% level, rats (strain
3 not specified; n = 30) exposed to 190 ppm for 5 minutes, 90 ppm for 15 minutes, or 72
4 ppm for 60 minutes showed signs of severe respiratory distress and eye irritation lasting
5 about two days; lung-to-body weight ratios were significantly increased during the first
6 48 hours after exposure. Pathological examination showed darkened areas of the lungs,
7 pulmonary edema, and an increased incidence of chronic murine pneumonia. Rats
8 exposed to 104 ppm for 5 minutes, 65 ppm for 15 minutes, or 28 ppm for 60 minutes
9 (about 25% of the LCsos) showed some respiratory distress or mild signs of nasal
10 irritation during exposure but lung-to-body weight ratios were increased only at the 104
11 and 65 ppm levels. No gross lesions were observed, but pulmonary edema was seen
12 microscopically. No adverse clinical signs of toxicity or pathological changes were
13 seen in rats exposed at 15% of the LCso (74 and 33 ppm for 5 and 15 minutes,
14 respectively).
15
16 Histological changes were examined in the lungs of male rats (strain and number
17 of animals not given) continuously exposed to 17 ppm NC>2 (Stephens et al. 1972).
18 After 2 hours of exposure, there was some precapillary and postcapillary engorgement
19 in the alveoli. Loss of cilia and occasional alveolar type I cell swelling were detectable
20 by 4 hours, the terminal bronchiolar epithelium had become uniform by 16 hours,
21 maximal macrophage numbers were reached by 24 hours, cellular hypertrophy had
22 begun by 48 hours, and mitotic figures became more prevalent in the epithelium of the
23 terminal bronchiole between 16 and 48 hours. Type I alveolar cells appeared to be the
24 most sensitive to NC>2 insult.
25
26 Results similar to those described above were obtained in a morphological study
27 of the Wistar rat lung (number of animals not given) following exposure to 20 ppm NC>2
28 for 20 hours (Hayashi et al. 1987). Cytoplasmic blebbing occurred in a small number of
29 type I cells immediately after exposure. Swelling and hyperplasia of type II cells and
30 pinocytotic vesicles of endothelial cells in capillaries followed by interstitial edema in the
31 alveolar walls were observed between days 5-15 postexposure. Twenty days after
32 exposure the lesions lessened and the lungs appeared normal after 35 days. Other studies
33 have confirmed alveolar and interstitial edema, bronchiolitis, bronchiolar epithelial cell
34 hyperplasia, loss of cilia, necrosis of type I cells, and/or type II cell hyperplasia 1-3 days
35 after exposure to 26 ppm NO2 for 24 hours (Schnizlein et al. 1980, Hillam et al. 1983) or
36 to 20 ppm for 24 hours (Rombout et al. 1986).
37
38 Long-Evans rats (number of animals not given) exposed to 20 ppm NO2 for up
39 to 24 hours showed minimal signs of irritation and changes in behavior. Microscopic
40 lesions were described as questionable evidence of lung congestion and interstitial
41 inflammation for up to 48 hours postexposure (Hine et al. 1970).
42
43 The effects of NO2 on the lung were compared between Sprague-Dawley neonatal
44 and adult rats (number of animals not given). Animals, 1-40 days old, were continuously
45 exposed to 14 ppm for 24, 48, or 72 hours. Prior to weaning (20 days old), exposure
46 resulted in only minor injury and loss of cilia from epithelial cells lining the terminal
32
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 airways. Subsequent to weaning, there was a progressive increase in lung injury with
2 maximum response reached at about 35 days of age (Stephens et al. 1978).
3
4 In a similar study to determine the sensitivity of adult and neonate animals to NO2
5 inhalation, Wistar rats (number of animals not given) aged 5-60 days were exposed
6 continuously for 3 days to 2 or 10 ppm (Azoulay-Dupuis et al. 1983). Litter exposures
7 prior to weaning included the dam. No clinical signs of toxicity or deaths were observed
8 in animals of any age except for body weight loss in dams of the 10 ppm-group. At 2
9 ppm, lung histopathology was normal in all animals. At 10 ppm in animals 45 days old
10 and older, fibrinous deposits were observed in the alveoli and the tracheal and
11 bronchiolar epithelia were occasionally devoid of cilia.
12
13 Alterations in lavage fluid have been assessed in male Long Evans rats (n = 6)
14 following exposure to 10, 20, 30, or 40 ppm for 4 hours. Cell-free lavage fluid contained
15 elevated lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate
16 dehydrogenase (IDH), glucose-6-phosphate dehydrogenase (GDH), acid phosphatase
17 (AP), and aryl sulfatase (AS) activity levels after exposure to >30 ppm. Total protein and
18 sialic acid were increased after exposure to >20 ppm. Protein and sialic acid
19 concentrations and AP activity level were similar to those in plasma indicating
20 transudation into the airways (Guth and Mavis 1985). The increases in LDH, MDH, and
21 GDH activity were significantly attenuated in animals maintained on diets providing
22 1000 mg/kg of a-tocopherol, suggesting that lipid peroxidation is involved in NO2
23 induced lung injury (Guth and Mavis 1986). Antioxidants in the lung were depleted, lipid
24 peroxidation products were elevated, and total cell count in BALF and alveolar
25 macrophage count were decreased while epithelial cell count was increased following
26 exposure of male Sprague-Dawley rats (n = 5) to 200 ppm for 15 minutes (Elsayed et al.
27 2002). Another study found changes in fatty acid composition of alveolar lavage
28 phospholipids following exposure of Wistar rats (n = 6) to 20 ppm NO2 for 12 hours
29 (Kobayashi et al. 1984). Increases in lavageable protein, polymorphonuclear
30 lymphocytes, and alveolar macrophages were also observed following exposure of male
31 Fischer 344 rats (n not given) to 100 ppm for 15 minutes (Lehnert et al. 1994).
32
33 Changes in minute ventilation, VE, were measured in male Fischer 344 rats (n =
34 12) following exposure to 100, 300, or 1000 ppm NO2 for 1-20 minutes (Lehnert et al.
35 1994). In general, reductions in VE were greater with the higher concentrations. For
36 example, reductions of about 7% and 15% were measured during 15- and 20-minute
37 exposure to 100 ppm, while reduction of about 20% and 28% were measured during 1-
38 and 2-minute exposures to 1000 ppm. Similarly, male Sprague-Dawley rats (n = 5)
39 exposed to 200 ppm for 15 minutes showed a decrease in minute ventilation that was due
40 to a decline in tidal volume but not in frequency of breathing (Elsayed et al. 2002).
41
42 Male Porton rats (n = 4) were exposed to an atmosphere of oxides of nitrogen
43 that was produced by mixing nitrogen dioxide and nitric oxide (Brown et al. 1983).
44 Exposures were to 518 ppm for 5 minutes or 1435 ppm for 1 minute. No clinical signs
45 of toxicity were observed during exposure to either concentration but "stertorous
46 respirations" appeared within 24 hours. Histologically, initial lung damage showed
33
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 thickening and blebbing of the alveolar epithelium followed by a latent period of about 6
2 hours after which development of edema of the interstitium and alveolar septum was
3 observed. The early changes were attributed to a direct oxidant effect.
4
5 Changes in lung immunity after NC>2 exposure have been described as increased
6 specific IgE, IgA, and IgG liters following exposure to 87 ppm for 1 hour (Siegel et al.
7 1997) or 5 ppm for 3 hours (Gilmour 1995), increased number of IgG anti-sheep red
8 blood cell antibody-forming cells in the lung-associated lymph nodes (Schnizlein et al.
9 1980), and cell proliferation in the spleen and thoracic lymph nodes (Hillam et al. 1983)
10 following exposure to 26 ppm for 24 hours.
11
12 3.2.9. Mice
13
14 Swiss-Webster mice (number of animals not given) exposed to 20 ppm NC>2 for
15 up to 24 hours showed minimal signs of irritation and changes in behavior.
16 Histologically, there was questionable evidence of lung congestion and interstitial
17 inflammation for up to 48 hours postexposure (Hine et al. 1970). The voluntary
18 running activity of mice on an activity-wheel was 80% and 17% of preexposure levels
19 during 6-hour exposures to 7.7 and 20.9 ppm, respectively (Murphy et al. 1964).
20
21 Female CD-I mice (n = 29-60) were examined for phenobarbital-induced
22 sleeping time after a 3 hour, whole body exposure to 0.125-5.0 ppm NC>2 (Miller et al.
23 1980). Sleeping time was significantly increased in animals exposed to >0.25 ppm
24 compared with air exposed controls. The authors stated that no effects in males were
25 observed until after three days of exposure (data not included). In contrast, the effect in
26 females decreased after the third day of exposure suggesting some tolerance may have
27 developed.
28
29 The alveolar septum from two female NMRI mice was examined
30 microscopically thirty-six hours after exposure to 35 ppm for 6 hours (Dillmann et al.
31 1967). Morphometric measurements found that the arithmetic mean thickness of the
32 alveoli was approximately 1.5x that of unexposed controls. No changes in the numbers
33 or types of cells present were observed and no interstitial edema was seen with
34 ultrastructure examination by electron microscopy.
35
36 Male CD-I mice (n = 5-9) were exposed to 50-140 ppm NC>2 for 1 hour and
37 biochemical and histological responses assessed immediately and 48-hours after exposure
38 (Siegel et al. 1989). Immediately after exposure to 140 ppm, cell death was visible in the
39 terminal bronchioles and there were significant increases in protease inhibitor activity,
40 pulmonary protein, and lung wet weight. Two days after exposure to 140 ppm, the
41 histological damage was exacerbated with complete obliteration of the alveolar structure,
42 progressive edema and congestion of the lungs, hypertrophy and hyperplasia of the
43 epithelial cells, and increased numbers of intraalveolar macrophages and neutrophils.
44 Also two days after exposure, there were dose-related increases in p-glucuronidase,
45 lactate dehydrogenase, and choline kinase activity as well as increased protease inhibitor
46 activity, pulmonary protein, and lung wet weight.
34
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1
2 To examine the effects of NC>2 on gaseous exchange in the lung, JCL:ICR mice (n
3 =6) were exposed to 5, 10, or 20 ppm for 24 hours (Suzuki et al. 1982). Significantly
4 increased lung wet weight and lung water content occurred at 10 and 20 ppm. In animals
5 exposed to 5 ppm, the gaseous exchange and metabolic rate of 62 and CO2 were
6 accelerated while in animals exposed to 10 and 20 ppm, gaseous exchange in the lung
7 was inhibited.
8
9 Continuous exposure of C56B1/6 mice (n = 60) to 20 ppm NC>2 for four days
10 resulted in significantly decreased food consumption and body weight, but no deaths
11 (Bouley etal. 1986).
12
13 3.3. Developmental/Reproductive Toxicity
14
15 The postnatal effects of prenatal exposure to NC>2 were investigated (Tabacova et
16 al. 1985). Pregnant Wistar rats (n = 20) were exposed to 0.265, 0.053, 0.53, or 5.3 ppm
17 NC>2 for 6 hours/day throughout pregnancy. Maternal effects were not reported or
18 discussed. Pup viability and body weight of the 5.3 ppm-group were significantly (p <
19 0.05) less than those of controls on lactation day 21. Exposure to >0.53 ppm resulted in
20 developmental delays and exposure to >0.053 ppm caused disturbances in neuromotor
21 development. Also at the two highest concentrations, hexobarbital sleeping time was
22 increased in the offspring and correlated with altered biochemical parameters in the
23 liver.
24
25 3.4. Genotoxicity
26
27 Three-week old male Sprague-Dawley rats were exposed by inhalation to 8, 15,
28 21, or 27 ppm NC>2 for 3 hours, maintained overnight before sacrifice, and lung cells
29 isolated. At concentrations of >15 ppm, there was a concentration-related increase in
30 mutation to ouabain resistance in lung cells. Concentration-dependent increases in
31 chromosome aberrations were observed at 8 and 27 ppm NC>2, the only concentrations
32 analyzed for aberrations (Isomura et al. 1984).
33
34 3.5. Subchronic and Chronic Toxicity/Carcinogenicity
35
36 Respiratory effects in humans from long-term environmental exposure to NC>2
37 have been discussed with the epidemiology studies in Section 2.2.2.
38
39 The effect of NC>2 on promotion of lung tumorigenesis induced by 7V-bis(2-
40 hydroxypropyl) nitrosamine (BHPN) was investigated in male Wistar rats (Ichinose et al.
41 1991). Animals were given a single intraperitoneal injection of 0.5 g BHPN/kg body
42 weight at 6 weeks of age and exposed to 0.04, 0.4, or 4.0 ppm NC>2 for 17 months. The
43 incidence of pulmonary tumors in rats exposed to BHPN plus 4 ppm NC>2 was 12.5%
44 (n.s.) with adenomas found in 4/40 rats (10%) and adenocarcinomas found in 1/40 rats
45 (2.5%). One adenoma was found in the control group (2.5%) and one in the 0.04 ppm
46 group, but none in the 0.4 ppm group. In addition, marked bronchiolar mucosal
35
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 hyperplasia was found in 17/40 rats (42.5%, p < 0.001) in the BHPN plus 4.0 ppm NO2
2 group.
3
4 3.6. Summary
5
6 Five- to 60-minute LCso values for NO2 in the rat ranged from 416 to 115 ppm,
7 respectively in one study (Carson et al. 1962) and from 833 to 168 ppm in another study
8 (Gray et al. 1954). The 15-minute LC50 for rabbits was 315 ppm (Carson et al. 1962). In
9 a study using varying concentration and duration of exposure, the first mortalities were
10 observed in dogs at 75 ppm for 4 hours, in rabbits at 75 ppm for 1 hour, in guinea pigs at
11 50 ppm for 1 hour, and in rats and mice at 50 ppm for 24 hours (Hine et al. 1970).
12 Histological alterations of the lungs following death included bronchiolitis, desquamated
13 bronchial epithelium, infiltration by polymorphonuclear cells, and edema. Enhanced
14 susceptibility to infection was shown in monkeys following exposure to 50 ppm for 2
15 hours (Henry et al. 1969) and in mice following exposure to 2 or 3.5 ppm for 3 hours
16 (Ehrlich 1978).
17
18 Pulmonary edema and histological alterations induced by exposure to NO2 have
19 been characterized in dogs, sheep, guinea pigs, hamsters, rats, and mice. Numerous
20 studies in rats have confirmed alveolar and interstitial edema, bronchiolitis, bronchiolar
21 epithelial cell hyperplasia, loss of cilia, necrosis of type I cells, and/or type II cell
22 hyperplasia 1-3 days after exposure to 26 ppm NO2 for 24 hours (Schnizlein et al. 1980;
23 Hillam et al. 1983) or to 20 ppm for 20 (Hayashi et al. 1987) or 24 hours (Rombout et
24 al. 1986).
25
26 Neonates appear less sensitive to NO2 than adult animals with progressive
27 increases in lung injury and deaths seen in older rats and guinea pigs (Stephens et al.
28 1978, Azoulay-Dupuis et al. 1983).
29
30 Only one study was found in which rats were exposed to N2O4 and pulmonary
31 lesions were similar to those described following NO2 exposure. No studies with
32 exposure to N2Os were found.
33
34 4. SPECIAL CONSIDERATIONS
35 4.1. Metabolism and Disposition
36
37 Total respiratory tract absorption of NO2 by humans exposed to 0.29-7.2 ppm for
38 <30 minutes during quiet respiration and during exercise has been measured at 81-90%
39 and 91-92%, respectively in healthy adults and 72% and 87%, respectively in asthmatics
40 (U.S. EPA 1993). In monkeys exposed to 0.30-0.91 ppm NO2 for <10 minutes, 50-60%
41 of the inspired gas has been shown to be retained during quiet respiration with
42 distribution throughout the lungs (Goldstein et al. 1977). While the isolated rat lung,
43 ventilated with 5 ppm for 90 minutes, retained 36% of the NO2 (Postlethwait and Mustafa
44 1981), the majority of labeled NO2 (exposure parameters not specified) was retained by
45 the upper respiratory tract of the rat (Russell et al. 1991).
46
36
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 Pulmonary absorption of NO2 has been studied using in vivo and in vitro models.
2 Uptake appears to be governed by the reaction between inhaled NO2 and constituents of
3 the pulmonary surface lining layer to form nitrite (Postlethwait and Bidani 1990, 1994,
4 Saul and Archer 1983). NO2 uptake is saturable with absorption proportional to inspired
5 dose (Postlethwait and Bidani 1994) and has been shown to increase as temperature
6 increases to a maximum of 10.6 jig NO2/min in an isolated lung model (Postlethwait and
7 Bidani 1990). The predominant reaction in the lungs involves hydrogen abstraction by
8 readily oxidizable tissue components such as proteins and lipids to form nitrous acid and
9 the nitrite radical (Postlethwait and Bidani 1994) and reaction with water to form nitrous
10 and nitric acids (Goldstein et al. 1977).
11
12 Distribution of inhaled NO2 or its metabolites is via the blood stream
13 (Goldstein et al. 1977). Nitrite formed in the lungs is oxidized to nitrate by
14 interactions with RBCs after diffusion into the vascular space (Postlethwait and
15 Mustafa 1981). Exposure of mice to 40 ppmNO2 produced slight (0.2%)
16 nitrosylhemoglobin but no methemoglobin (Oda et al. 1980) and an increase in both
17 nitrite and nitrate that reached equilibrium in 10 and 30 minutes, respectively (Oda et
18 al. 1981). After cessation of exposure, nitrite had a half-life of several minutes and
19 nitrate had a half-life of about 1 hour (Oda et al. 1981). Urinary excretion of nitrate
20 has been shown to be linearly related to the NO2 concentration administered via
21 inhalation (Saul and Archer 1983).
22
23 4.2. Mechanism of Toxicity
24
25 NO2 is an irritant to the mucous membranes and may cause coughing and dyspnea
26 during exposure. After less severe exposure, symptoms may persist for several hours
27 before subsiding (NIOSH 1976). With more severe exposure, pulmonary edema ensues
28 with signs of chest pain, cough, dyspnea, cyanosis, and moist rales heard on auscultation
29 (NIOSH 1976, Douglas et al. 1989). Death from NO2 inhalation is caused by
30 bronchospasm and pulmonary edema in association with hypoxemia and respiratory
31 acidosis, metabolic acidosis, shift of the oxyhemoglobin dissociation curve to the left,
32 and arterial hypotension (Douglas et al. 1989). A characteristic of NO2 intoxication after
33 the acute phase is a period of apparent recovery followed by late-onset bronchiolar injury
34 that manifests as bronchiolitis fibrosa obliterans (NIOSH 1976, NRC 1977, Hamilton
35 1983, Douglas et al. 1989).
36
37 Toxicity from acute exposure can be described in one of three categories: 1)
38 immediate death after very heavy exposure, 2) delayed symptoms with development of
39 edema within 48 hours, and 3) apparent recovery from immediate effects but later chronic
40 chest disease of varying severity (NRC 1977, Hamilton 1983). Morphological and
41 biochemical changes in the lungs during these phases were studied in mice exposed to
42 140 ppm for 1 hour (Siegel et al. 1989). Immediately after exposure, cell death was
43 noted in areas adjacent to the distal terminal bronchioles, and protease inhibitor activity,
44 lung protein content, and lung wet weight were significantly elevated. Two days after
45 exposure, the histological damage was exacerbated with complete obliteration of the
46 alvoelar structure, progressive edema and congestion of the lungs, hypertrophy and
37
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 hyperplasia of the epithelial cells, increased numbers of intraalveolar macrophages and
2 neutrophils. Also two days after exposure, there were dose-related increases in P-
3 glucuronidase, lactate dehydrogenase, and choline kinase activity as well as increased
4 protease inhibitor activity, pulmonary protein, and lung wet weight. Pulmonary injury is
5 characterized by loss of ciliated cells, disruption of tight capillary junctions, degeneration
6 of type I cells, and proliferation of type II cells (Siegel et al. 1989, Elsayed 1994).
7
8 The predominant reaction in the lungs involves hydrogen abstraction by readily
9 oxidizable tissue components such as proteins and lipids to form nitrous acid and the
10 nitrite radical (Postlethwait and Bidani 1994 U.S. EPA 1995) and reaction with water to
11 form nitrous and nitric acids (Greenbaum et al. 1967, Goldstein et al. 1977). This
12 reaction can lead to one mechanism by which NO2 causes pulmonary injury, lipid
13 peroxidation. NO2 is a free radical that can attack unsaturated fatty acids in the cell
14 membrane forming carbon and oxygen centered radicals in a chain reaction (Ainslie
15 1993, Elsayed 1994, U.S. EPA 1995). This hypothesis is supported by studies on the
16 effects of antioxidants on NO2 exposure in humans and animals. Four-week
17 supplementation with vitamins C and E before exposure to 4 ppm for 3 hours resulted in
18 a marked decrease in the amount of conjugated dienes and attenuated the decrease in
19 elastase activity inhibitory capacity in the alveolar lining fluid of healthy, human
20 volunteers (Mohsenin 1991). Guinea pigs maintained on an ascorbic acid-deficient diet
21 had increased lung lavage fluid protein following exposure to 4.8 ppm NO2 for 3 hours
22 and increased wet lung weight, increased nonprotein sulfhydryl and ascorbic acid content
23 of the lungs, and decreased a-tocopherol content of the lungs following exposure to 4.5
24 ppm for 16 hours. These changes were not seen in animals maintained on normal guinea
25 pig diets (Hatch et al. 1986). Rats exposed to 30 and 40 ppm for 4 hours had elevations
26 of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and glucose-6-
27 phosphate dehydrogenase (GDH) activity in lavage fluid which were significantly
28 attenuated in animals maintained on diets providing 1000 mg/kg of a-tocopherol (Guth
29 and Mavis 1986). Another study found changes in fatty acid composition of alveolar
30 lavage phospholipids following exposure of rats to 10 ppm NO2 for 12 hours (Kobayashi
31 etal. 1984).
32
33 4.3. Oxides of Nitrogen
34
35 NO2 exists as an equilibrium mixture of NO2 and N2C>4 but the dimer is not
36 important at ambient concentrations (U.S. EPA 1993). The two compounds are phase-
37 related forms with N2C>4 favored in the liquid phase and NO2 favored in the gaseous
38 phase. An equilibrium distribution is reached which favors the lowest energy state in
39 the phase. As a result when N2C>4 is released it vaporizes and dissociates into NO2,
40 making it nearly impossible to generate a significant concentration of N2C>4 at
41 atmospheric pressure and ambient temperature, without generating a vastly higher
42 concentration of NO2. A rate for this reaction was not found, and because of this effect,
43 almost no inhalation toxicity data are available on N2C>4. No information was found on
44 the interactions of N2Os.
45
46 Another oxide of nitrogen, nitric oxide (NO), is unstable in air and undergoes
38
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 spontaneous oxidation to NC>2. If the exposure concentration of NO is not high enough
2 to be lethal due to methemoglobin formation, the victim can recover completely. On
3 the other hand, concentrations of NO2 that are not rapidly lethal may cause persistent
4 effects and in some cases cause death from pulmonary edema after a delay of several
5 days (NIOSH 1976). In photochemical smog, NO2 absorbs sunlight of wavelengths
6 between 290-430 nm and decomposes to NO and O (U.S. EPA 1993). If NO is of
7 concern, reference should be made to the AEGL technical support document for nitric
8 oxide.
9
10 4.4. Other Relevant Information
11 4.4.1. Species Variability
12
13 Several studies indicate that there is a size-dependent species sensitivity to NO2
14 with larger animals apparently less sensitive than smaller animals such as rodents. Dogs
15 showed only mild signs of irritation at concentrations that caused pulmonary edema in
16 rats (Carson et al. 1962). Dogs also survived exposures to 1000 ppm for 136 minutes and
17 5000 ppm for up to 22 minutes (Greenbaum et al. 1967) and sheep survived exposure to
18 500 ppm for 15-20 minutes (Januszkiewicz and Mayorga 1994). In contrast, 15-minute
19 and 1-hour LCso values in the rat ranged from 201-420 ppm and 115-168 ppm,
20 respectively (Gray et al. 1954, Carson et al. 1962). Based on the data available, humans
21 are not more sensitive than larger laboratory animals. For example, irritation was
22 reported for humans exposed to 30 ppm for 2 hours (Henschler et al. 1960), in dogs
23 exposed to 20 ppm for 24 hours (Hine et al. 1970), and in monkeys exposed to 35 ppm
24 for 2 hours (Henry et al. 1969).
25
26 Elsayed et al. (2002) examined species variability through dosimetry; the
27 calculated total inspired dose from experimental measurements in rats and sheep was
28 compared to the theoretical dose of an average human. Whether normalized for body
29 weight, lung volume, or alveolar surface area, the total effective dose was rats » sheep >
30 humans. Taking physiologic and anatomical factors into consideration, rats had a much
31 higher effective dose than the larger animals. The authors concluded that NO2 toxicity is
32 associated with inhaled-dose distribution per unit lung volume or lung surface rather than
33 per unit body mass (Elsayed et al. 2002).
34
35 4.4.2. Susceptible Populations
36
37 For chronic, low-level exposures, U.S. EPA (1995) has identified two
38 populations as potentially at risk from NO2 exposure: children ages 5-12 and persons
39 with pre-existing respiratory disease. Conclusions drawn from epidemiology studies
40 were that children ages 5-12 years old had an increased risk of about 20% for
41 developing respiratory symptoms and disease with each increase of 0.015 ppm in
42 estimated 2-week average NO2 exposure (mean weekly concentrations in bedrooms
43 0.008-0.065 ppm) and that no evidence for increased risk was found for infants <2 years
44 old. This conclusion is supported somewhat by animal data in which adult animals were
45 more sensitive than neonates to the effects of NO2 (Azoulay-Dupuis et al. 1983,
46 Stephens et al. 1978). Reduced ventilatory reserves may prevent individuals with
39
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 respiratory disease from resuming normal activity following exposure to NO2 (U.S. EPA
2 1995). However, it is not certain whether these populations are also at particular risk
3 from acute exposure scenarios.
4
5 Taken together, the data summarized in section 2.2.3 indicate that some
6 asthmatics exposed to 0.3-0.5 ppm NO2 may respond with either subjective symptoms or
7 slight changes in pulmonary function of no clinical significance. At approximately these
8 same concentrations of NO2, subsequent exposure of asthmatics to an agent that causes
9 non-specific airway responsiveness resulted in slight hyperreactivity, but the response is
10 not more severe than to NO2 alone (e.g. while some asthmatics respond to a bronchial
11 challenge and to NO2, the response to the challenge is not additively increased from prior
12 exposure to NO2). In contrast, some asthmatics did not respond to NO2 with changes in
13 pulmonary function or symptoms at concentrations of 0.5-4 ppm. The responses of
14 healthy individuals to NO2 exposures are also variable, with some, but not all, having
15 slight changes in pulmonary function following exposure to 5 ppm. All reported
16 responses in both asthmatic and healthy subjects at the concentrations discussed were
17 slight and of questionable biological or clinical significance.
18
19 Conclusions regarding differences in susceptibility between healthy and asthmatic
20 individuals are difficult to draw from the available data because of the high variability in
21 responses among both groups. There is only one study which has measured the responses
22 of both healthy and asthmatic individuals with the same study protocol (Linn and
23 Hackney 1983). Dose-response patterns are not discernable at these low concentrations
24 and clear thresholds are not apparent. Some individuals reported clinical symptoms in
25 the absence of changes in pulmonary function, while other individuals had measurable
26 changes in pulmonary function tests but no symptoms. One proposed explanation for the
27 variability in the responses of asthmatics to inhaled NO2 is the existence of a subgroup of
28 "responders." From one laboratory, several asthmatics were identified as equally
29 responsive to 0.3 ppm in more than one study (Bauer et al. 1985, 1986). However, the
30 investigators could find no common identifiers for these "responders" such as degree of
31 baseline obstruction or their inherent airway reactivity to carbachol or cold air (Utell
32 1989). Although some individuals had a measurable response at lower concentrations,
33 the magnitude of the reported changes was not biologically or clinically significant in
34 either asthmatics or healthy individuals.
35
36 4.4.3. Concentration-Response Relationship
37
38 As discussed below for AEGL-2 and -3 levels, extrapolations were made to each
39 of the time points using Cn x t = k where n = 3.5 (ten Berge et al. 1986). The value of n
40 was calculated by ten Berge et al. based on the data of Hine et al. (1970). The large value
41 of n indicates that concentration is more important than duration for the effects of
42 exposure to NO2. Support for this supposition also comes from Gardner et al. (1979)
43 who showed that short-term exposure to high concentrations resulted in greater effects (as
44 measured by mortality in an infectivity model in mice) than exposure to lower
45 concentrations administered over a longer duration.
46
40
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 4.4.4. Susceptibility to infection
2
3 To determine the effects of NC>2 on resistance to infection, squirrel monkeys were
4 challenged with Klebsiellapneumoniae within 24 hour after exposure. No deaths
5 occurred from exposure to NC>2 alone, however, 3/3 monkeys died within 72 hours after
6 50 ppm NC>2 exposure for 2 hours followed by K. pneumoniae challenge; massive
7 infection was present in the lungs and other organs. Exposure of monkeys to 10 ppm for
8 2 hours followed by K. pneumoniae challenge 3-5 days later did not result in death of the
9 animals, but bacteria were still present in lung tissue at necropsy up to 46 days after
10 challenge indicating reduced clearence (Henry et al. 1969).
11
12 Numerous studies have reported enhanced susceptibility of mice to infectious
13 agents following exposure to NC>2. Most of these studies have been reviewed by U.S.
14 EPA (1993) and only a few are described here. Gardner et al. (1977) demonstrated that
15 the concentration-time relationship was linear for 20% mortality using an infectivity
16 model in mice challenged with Streptococcuspyogenes; NC>2 exposures ranged from
17 0.5-28 ppm for 6 minutes to 12 months. Similarly, mortality was increased in mice
18 challenged with S. pyogenes in response to short-term exposure to a high concentration
19 of NC>2 compared to a lower concentration administered over a longer duration when
20 the concentration x time product was held constant. A single 3-hour exposure to 2.0 or
21 3.5 ppm NC>2 enhanced the susceptibility of three strains of mice to streptococcal
22 pneumonia and influenza infection as seen by excess mortality and reduced survival
23 time (Ehrlich 1978). Mice exercised in a motorized wheel during exposure to 3 ppm
24 for 3 hours followed by challenge with S. pyogenes had significantly increased
25 mortality compared to non-exercised animals (Tiling et al. 1980). Pulmonary bacterial
26 defenses against Staphylococcus aureus were suppressed following exposure of Swiss
27 mice to concentrations of >4 ppm for 4 hours (Jakab 1987). Significantly decreased
28 pulmonary bactericidal activity was shown in Swiss mice infected with S. aureus then
29 exposed to 7, 9.2, or 14.8 ppm NC>2 for 4 hours, or exposed to 2.3 or 6.6 ppm for 17
30 hours prior to infection. Histologically the lungs of mice exposed to >9.2 ppm for 4
31 hours showed vascular hyperemia while those from mice exposed to >2.3 ppm for 17
32 hours had minor vascular hyperemia and interstitial edema (Goldstein et al. 1973).
33 Enhanced susceptibility to infection was observed in CD-I mice exposed to 5 ppm for 6
34 hours/day on two consecutive days prior to inoculation with murine cytomegalovirus,
35 followed by exposure to 5 ppm NC>2 for 6 hours/day for four consecutive days; there
36 was no histological evidence of lung injury (Rose et al. 1989). Continuous exposure of
37 mice to 20 ppm NC>2 for 4 days resulted in impairment of acquired resistance
38 (decreased EDso) of C57B1/6 mice immunized prior to Klebsiella pneumoniae
39 challenge (Bouley etal. 1986).
40
41 Alterations in host defense mechanisms have been demonstrated in rabbits. Male
42 and female New Zealand rabbits exposed for 3 hours to varying concentrations of NC>2
43 had an increase in polymorphonuclear leukocytes obtained by pulmonary lavage at >8
44 ppm with the peak infiltration 6-9 hours after the end of exposure (Gardner et al. 1969).
45 In other experiments, these authors demonstrated that the response persisted up to 72
46 hours post exposure and that phagocytic activity was inhibited.
41
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1
2 Mice have also been used extensively as a model for immune function
3 alterations following NC>2 exposure. Decreases in splenic and thymic weights,
4 cellularity, plaque-forming cell (PFC) responses, and hemagglutinins (HA), along with
5 decreased body weight, were observed in C56B1/6 mice exposed to 20 ppm NC>2 for 48
6 hours (Azoulay-Dupuis et al. 1985). Significant suppression of primary antibody
7 responses (HA and PFC) were also seen following exposure of BALB/c mice to 20 or
8 40 ppm for 12 hours (Hidekazu and Fujio 1981). Phytohemagglutinin and bacterial
9 lipopolysaccharide responses were depressed in mice exposed continuously to 0.5 ppm
10 or to 0.1 ppm with daily 3-h peaks (5 days per week) of either 0.25 ppm, O.Sppm, or 1.0
11 ppm (Maigetter et al. 1978). Other effects of NC>2 on cellular and humoral immunity
12 have been reviewed by U.S. EPA (1993) but are not relevant to derivation of AEGL
13 values.
14
15 5. DATA ANALYSIS FOR AEGL-1
16
17 AEGL-1 is the airborne concentration (expressed as parts per million or
18 milligrams per cubic meter [ppm or mg/m3]) of a substance above which it is predicted
19 that the general population, including susceptible individuals, could experience notable
20 discomfort, irritation, or certain asymptomatic, non-sensory effects. The effects are not
21 disabling and are transient and reversible upon cessation of exposure.
22
23 5.1. Summary of Human Data Relevant to AEGL-1
24
25 The evidence indicates that some asthmatics exposed to 0.3-0.5 ppm NC>2 may
26 respond with either subjective symptoms or slight changes in pulmonary function of no
27 clinical significance. At approximately these same concentrations of NC>2 some
28 asthmatics may show slight hyperreactivity to a bronchial challenge, but the response is
29 no more severe than the response to NC>2 alone (e.g. while some asthmatics respond to a
30 bronchial challenge and to NC>2, the response to the challenge is not additively increased
31 from prior exposure to NC>2). In contrast, some asthmatics did not respond to NC>2 at
32 concentrations of 0.5-4 ppm. The responses of healthy individuals to NC>2 exposures are
33 also variable, with some, but not all, responding to 5 ppm.
34
35 Kerr et al. (1978, 1979) reported that 7/13 asthmatics experienced slight burning
36 of the eyes, slight headache, chest tightness, or labored breathing with exercise during
37 exposure to 0.5 ppm for 2 hours; at this concentration the odor of NC>2 was perceptible
38 but the subjects became unaware of it after about 15 minutes. No changes in any
39 pulmonary function tests were found immediately following the chamber exposure (Kerr
40 et al. 1978, 1979). Significant group mean reductions in FEVi (-17.3% vs -10.0%) and
41 specific airway conductance (-13.5% vs -8.5%) occurred in asthmatics after exercise
42 during exposure to 0.3 ppm for 4 hours and 1/6 individuals experienced chest tightness
43 and wheezing (Bauer et al. 1985). The onset of effects was delayed when exposures were
44 by oral-nasal inhalation compared with oral inhalation. This delay may result from
45 scrubbing within the upper airway. In a similar study, asthmatics exposed to 0.3 ppm for
46 30 minutes at rest followed by 10 minutes of exercise had significantly greater reductions
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
in FEVi (10% vs 4% with air) and partial expiratory flow rates at 60% of total lung
capacity, but no symptoms were reported (Bauer et al. 1986). In a preliminary study with
13 asthmatics exposed to 0.3 ppm for 110 minutes, slight cough and dry mouth and throat
and significantly greater reduction (11% vs 7%) in FEVi occurred after exercise,
however, in a larger study, no changes in pulmonary function were measured and no
symptoms were reported following exposure of 21 asthmatics to concentrations up to 0.6
ppm for 75 minutes (Roger et al. 1990). The mean drop in FEVi for asthmatics during a
3-hour exposure to 1 ppm NO2 (2.5%) with intermittent exercise was significantly greater
than the drop during air (1.3%) exposure with intermittent exercise; in BALF, levels of 6-
keto-prostaglandinia were decreased and levels of thromboxane B2 and prostaglandin D2
were increased after NO2 exposure (Torres et al. 1995).
5.2. Summary of Animal Data Relevant to AEGL-1
Animal data relevant to derivation of AEGL-1 are limited. Slight irritation was
noted in squirrel monkeys exposed to 10 and 15 ppm for 2 hours (Henry et al. 1969)
and mild sensory effects occurred in dogs exposed to 125 ppm for 5 minutes, 52 ppm
for 15 minutes, or 39 ppm for 60 minutes (Carson et al. 1962).
5.3. Derivation of AEGL-1
The study by Kerr et al. (1978, 1979) was considered the most appropriate to use
as the basis for AEGL-1 values. Exposure of asthmatics to 0.5 ppm NO2 for 2 hours
resulted in clinical signs but no changes in pulmonary function. Since asthmatics are
potentially the most susceptible population, no uncertainty factor was applied. Therefore,
a concentration of 0.94 mg/m3 (0.50 ppm NO2 or 0.25 ppm N2C>4) was adopted for all
time points (Table 5) because adaptation to mild sensory irritation occurs. In addition,
animal responses to NO2 exposure have demonstrated a much greater dependence upon
concentration than upon time; therefore, extending the 2-hour concentration to 8 hours
should not exacerbate the human response.
TABLE 5: AEGL-1 Values for Nitrogen Dioxide and Nitrogen Tetroxide
Chemical
N02
N204
10-minute
0.94 mg/m3
(0.50 ppm)
0.94 mg/m3
(0.25 ppm)
30-minute
0.94 mg/m3
(0.50 ppm)
0.94 mg/m3
(0.25 ppm)
1-hour
0.94 mg/m3
(0.50 ppm)
0.94 mg/m3
(0.25 ppm)
4-hour
0.94 mg/m3
(0.50 ppm)
0.94 mg/m3
(0.25 ppm)
8 -hour
0.94 mg/m3
(0.50 ppm)
0.94 mg/m3
(0.25 ppm)
32
33
34
35
36
37
38
39
6. DATA ANALYSIS FOR AEGL-2
AEGL-2 is the airborne concentration (expressed as ppm or mg/m3) of a
substance above which it is predicted that the general population, including susceptible
individuals, could experience irreversible or other serious, long-lasting adverse health
effects or an impaired ability to escape.
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1
2 6.1. Summary of Human Data Relevant to AEGL-2
3
4 Human data relevant to AEGL-2 are limited but consistent. Henschler et al.
5 (1960) performed several experiments on healthy, male volunteers and found that
6 exposure to 30 ppm for 2 hours caused definite discomfort. Three individuals exposed to
7 30 ppm for 2 hours perceived an intense odor upon entering the chamber, the odor
8 quickly diminished and was completely absent after 25-40 minutes. One individual
9 experienced a slight tickling of the nose and throat mucous membranes after 30 minutes,
10 the two others after 40 minutes. From 70 minutes on, all subjects experienced a burning
11 sensation and an increasingly severe cough for the next 10-20 minutes, but coughing
12 decreased from 100 minutes on. However, the burning sensation continued and moved
13 into the lower sections of the airways and was finally felt deep in the chest. At this time,
14 marked sputum secretion and dyspnea were noted. Toward the end of the exposure, the
15 subjects condition was described as bothersome and barely tolerable. A sensation of
16 pressure and increased sputum secretion continued for several hours after cessation of
17 exposure (Henschler et al. 1960). In a similar experiment (Henschler and Liitke 1963)
18 groups of 4 or 8 healthy male volunteers were exposed to 10 ppm for 6 hours or to 20
19 ppm for 2 hours. All subjects, upon entering the chamber, noted the odor which
20 diminished rapidly. At 20 ppm minor scratchiness of the throat was felt after about 50
21 minutes and 3/8 experienced slight headaches towards the end of the exposure period.
22
23 6.2. Summary of Animal Data Relevant to AEGL-2
24
25 Several animal studies are relevant to AEGL-2 derivation. Hine et al. (1970)
26 noted lacrimation, reddening of the conjunctivae, and increased respiration in 5 species
27 exposed to >40 ppm for varying durations. Lethality did not occur until concentrations
28 and durations reached 75 ppm for 4 hours in the dog and 1 hour in the rabbit, 50 ppm for
29 1 hour in the guinea pig, and 50 ppm for 24 hours in the rat and mouse. At 20 ppm for 24
30 hours, all species showed minimal signs of irritation and changes in behavior with
31 histopathological lesions described as questionable evidence of lung congestion and
32 interstitial inflammation.
33
34 Exposure of monkeys to 35 ppm for two hours resulted in irritation as measured
35 by changes in lung function and microscopic lesions in the lung (Henry et al. 1969).
36 The histological lesions in the lung were characterized by Siegel et al. (1989) following
37 exposure of mice to 140 ppm for 1 hour. Carson et al. (1962) conducted a series of
38 experiments in dogs and rats. Mild irritation and some respiratory effects, but no gross
39 or microscopic lesions, were noted in dogs exposed to 53 or 39 ppm for 1 hour while
40 rats exposed to 72 ppm for 1 hour showed signs of severe respiratory distress and eye
41 irritation as well as gross lesions in the lung and evidence of infection.
42
43 Developmental delays and disturbances in neuromotor development were reported
44 for rat pups following maternal exposure (Tabacova et al. 1985). However, these effects
45 were reported to have occurred at levels near ambient concentrations and are well below
46 those of most other studies in both humans and animals.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
6.3. Derivation of AEGL-2
Based on both human and animal data, it appears that a concentration of >30 ppm
NC>2 is required before marked irritation, discomfort, and respiratory effects occur.
Therefore, a concentration of 30 ppm for a 2-hour exposure of humans (Henschler et al.
1960) was used to derive AEGL-2 values. The point of departure is considered a
threshold for AEGL-2 since the effects noted by the subjects would not impair the ability
to escape and the effects were reversible after cessation of exposure. Values scaled for
the derivation of the 10- and 30-minute and 1-, 4-, and 8-hour AEGL-2 endpoints were
calculated from Cn x t = k using n = 3.5 (ten Berge et al. 1986). The value of n was
calculated by ten Berge et al. from the data of all species together from Hine et al. (1970).
An intraspecies uncertainty factor of 3 was applied to account for sensitive
subpopulations because the mechanism of action of a direct acting irritant is not expected
to differ greatly among individuals. With additional uncertainty factors the values would
be inconsistent with some of the experimental data for asthmatics, such as the no-
adverse-effect concentration of 4 ppm in the study by Linn and Hackney (1984). AEGL-
2 values are presented in Table 6.
TABLE 6: AEGL-2 Values for Nitrogen Dioxide and Nitrogen Tetroxide
Chemical
N02
N204
10-minute
38 mg/m3
(20 ppm)
38 mg/m3
(10 ppm)
30-minute
28 mg/m3
(15 ppm)
28 mg/m3
(7.6 ppm)
1-hour
23 mg/m3
(12 ppm)
23 mg/m3
(6.2 ppm)
4-hour
15 mg/m3
(8.2 ppm)
15 mg/m
(4.1 ppm)
8 -hour
13 mg/m3
(6.7 ppm)
13 mg/m
(3. 5 ppm)
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
These levels are not expected to cause severe effects as coal miners were exposed
to peakNO2 concentrations of 14 ppm without adverse consequences (Robertson et al.
1984) and it can be assumed that the peak levels were not sustained longer than a few
minutes. Similar AEGL-2 values are derived using the exposure of 140 ppm for 1 hour
in the mouse (Siegel et al. 1989) and an uncertainty factor of 10 or the exposure of 35
ppm for 2 hours in the monkey (Henry et al. 1969) and an uncertainty factor of 3. If the
animal data from either Hine et al. (1970) or Carson et al. (1962) are used for the basis of
derivation, the AEGL-2 values are even more conservative than those derived with the
use of human data.
7. DATA ANALYSIS FOR AEGL-3
AEGL-3 is the airborne concentration (expressed as ppm or mg/m3) of a
substance above which it is predicted that the general population, including susceptible
individuals, could experience life-threatening health effects or death.
7.1. Summary of Human Data Relevant to AEGL-3
A welder was hospitalized with pulmonary edema after exposure to
45
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 approximately 90 ppm for 30-40 minutes (Norwood et al. 1966). It is possible that
2 without medical intervention, the exposure could have been fatal.
3
4 Concentrations of NO2 above 150 ppm are probably fatal to humans due to
5 bronchospasm and pulmonary edema (NRC 1977, Douglas et al. 1989). A human 1-hour
6 LCso of 174 ppm was estimated from data in 5 animal species (Book 1982), however,
7 these are not considered valid experimental data on which to base AEGL-3. No other
8 human data were relevant to derivation of AEGL-3.
9
10 7.2. Summary of Animal Data Relevant to AEGL-3
11
12 Squirrel monkeys (n = 2-6/group) were exposed to 10-50 ppm NO2 for 2 hours
13 with respiratory function monitored during exposure (Henry et al. 1969). NO2 exposure
14 alone resulted in a markedly increased respiratory rate and decreased tidal volume
15 during exposures to 50 or 35 ppm, but caused only slight effects at 15 and 10 ppm. Mild
16 histopathological changes in the lungs were noted after exposure to 10 and 15 ppm,
17 however, marked changes in lung structure were observed after exposure to 35 and 50
18 ppm. At 35 ppm, areas of the lung were collapsed with basophilic alveolar septa, in
19 other areas the alveoli were expanded with septal wall thinning, and the bronchi were
20 moderately inflamed with some proliferation of the surface epithelium. At 50 ppm,
21 extreme vesicular dilatation or total collapse of alveoli was observed, lymphocyte
22 infiltration was seen with extensive edema, and surface erosion of the epithelium of the
23 bronchi was observed. In addition to the effects on the lungs, interstitial fibrosis (35
24 ppm) and edema (50 ppm) of cardiac tissue, glomerular tuft swelling in the kidney (35
25 and 50 ppm), lymphocyte infiltration in the kidney and liver (50 ppm), and congestion
26 and centrilobular necrosis in the liver (50 ppm) were observed.
27
28 Rats exposed to 72 ppm for 60 minutes (approximately 50% of the LD50) showed
29 signs of severe respiratory distress and eye irritation lasting about 2 days; lung-to-body
30 weight ratios were significantly increased during the first 48 hours after exposure (Carson
31 etal. 1962).
32
33 Lethality in 5 animal species first occurred at exposure concentrations and
34 durations of 75 ppm for 4 hours in the dog and 1 hour in the rabbit, 50 ppm for 1 hour
35 in the guinea pig, and 50 ppm for 24 hours in the rat and mouse (Hine et al. 1970). In
36 general, the larger animals, including humans, are less susceptible to toxicity from NO2
37 inhalation than are the rodents.
38
39 7.3. Derivation of AEGL-3
40
41 The data from the monkey are considered the best available for derivation of
42 AEGL-3 values. Signs of marked irritation and severe lung histopathology were observed
43 from exposure to 50 ppm for 2 hours. This exposure scenario was extrapolated to the 10-
44 and 30-minute and 1-, 4-, and 8-hour time points using the equation Cn x t = k where n =
45 3.5 (ten Berge et al. 1986). The value of n was calculated by ten Berge et al. from the
46 data of all species together from Hine et al. (1970). A total uncertainty factor of 3 was
46
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applied which includes a 3 for intraspecies variability and a 1 for interspecies variability.
Use of a greater intraspecies uncertainty factor was not considered necessary because the
mechanism of action is not expected to differ greatly among individuals. Because the
endpoint in the monkey study is below the definition of AEGL-3, human data support the
point of departure and derived values, and due to the similarities of the respiratory tract
between humans and monkeys, additional interspecies uncertainty factors are not
considered necessary. The mechanism of action of NO2 does not vary between species
with the target at the alveoli. AEGL-3 values for NO2 and N2O4 are listed in Table 7.
TABLE 7: AEGL-3 Values for Nitrogen Dioxide and Nitrogen Tetroxide
Chemical
NO2
N2O4
10-minute
64 mg/m
(34 ppm)
64 mg/m3
(17 ppm)
30-minute
47 mg/m3
(25 ppm)
47 mg/m3
(13 ppm)
1-hour
38 mg/m3
(20 ppm)
38 mg/m3
(10 ppm)
4-hour
26 mg/m3
(14 ppm)
26 mg/m3
(7.0 ppm)
8 -hour
21 mg/m3
(1 1 ppm)
21 mg/m3
(5. 7 ppm)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
The AEGL-3 values are supported by human data from the welder. Pulmonary
edema, confirmed on X-ray, resulted from exposure to approximately 90 ppm for up to
40 minutes (Norwood et al. 1966). If this exposure scenario is used for derivation of
AEGL-3 values with an uncertainty factor of 3 the 10- and 30-minute and 1-, 4-, and 8-
hour values are 45, 33, 27, 18, and 15 ppm, respectively. Similar results are obtained
using the exposure of rats to 72 ppm for 1 hour (Carson et al. 1962) and an uncertainty
factor of 3. In addition, the AEGL-3 values are below the concentrations at which
lethality first occurred in five animal species (Hine et al. 1970).
8. SUMMARY OF AEGLS
8.1. AEGL Values and Toxicity Endpoints
The derived AEGL values for various levels of effect and durations of exposure
are summarized in Tables 8 and 9. Values were derived based on data from human and
animal exposures to NO2 and are considered applicable to the other oxides of nitrogen.
Values for N2O4 in units of ppm have been calculated on a molar basis as presented
below.
TABLE 8: Summary of AEGL Values for Nitrogen Dioxide (mg/m3 [ppm])
AEGL Level
AEGL-1
AEGL-2
AEGL-3
10-minute
0.94 (0.50)
38 (20)
64 (34)
30-minute
0.94 (0.50)
28(15)
47 (25)
1-hour
0.94 (0.50)
23 (12)
38 (20)
4-hour
0.94 (0.50)
15 (8.2)
26 (14)
8 -hour
0.94 (0.50)
13 (6.7)
21(11)
30
31
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TABLE 9: Summary of AEGL Values for Nitrogen Tetroxide (mg/m3 [ppm])
AEGL Level
AEGL-1
AEGL-2
AEGL-3
10-minute
0.94 (0.25)
38(10)
64 (17)
30-minute
0.94 (0.25)
28 (7.6)
47(13)
1-hour
0.94 (0.25)
23 (6.2)
38(10)
4-hour
0.94 (0.25)
15(4.1)
26 (7.0)
8 -hour
0.94 (0.25)
13 (3.5)
21 (5.7)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
8.2. Comparison with Other Standards and Criteria
Standards and guidance levels for workplace and community exposures to NC>2
are listed in Table 10. No standards or guidance for exposure to N2O4 were found. The
ACGffl recommends a TLV of 3 ppm for workers (ACGffl 2003) while the OSHA PEL
is a ceiling of 5 ppm (OSHA 1999). The NIOSH IDLH is 20 ppm (NIOSH 1996) which
is exactly between the 30-minute AEGL-2 and AEGL-3 values. The IDLH is reported as
based on acute inhalation data in humans, but no primary references were listed in the
documentation; NIOSH notes that the IDLH may be a conservative value due to the lack
of relevant acute toxicity data for workers exposed to concentrations above 20 ppm.
ERPGs (AIHA 2003), based on human and animal data, are similar to the 1-hour AEGL
values. The NRC's 1-hour EEGL is 1 ppm (NRC 1985) for workplace conditions. The
occupational exposure limits from ACGIH, Germany, The Netherlands, and Sweden are
2-5 ppm.
In addition to the standards listed in Table 10, air quality standards have also
been developed for NO2. The National Ambient Air Quality Standard is 0.053 ppm
(U.S. EPA 1997) with Significant Harm Levels of 2 ppm for a 1-hour average and 0.5
ppm for a 24-hour average (U.S. EPA 1987a). The Level of Concern is 5 ppm (U.S.
EPA 1987b). The state of California has adopted 0.25 ppm as the standard for a 1-hour
exposure to protect sensitive individuals (Cal. EPA 1992).
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TABLE 10: Extant Standards and Guidelines for Nitrogen Dioxide
Guideline
AEGL-1
AEGL-2
AEGL-3
ERPG-1 (AfflA)3
ERPG-2 (AfflA)
ERPG-3 (AfflA)
EEGL (NRC)b
IDLH (NIOSH)C
REL-STEL (NIOSH)d
PEL-STEL (OSHA)e
PEL-TWA (OSHA)f
TLV-TWA
(ACGIH)g
TLV-STEL
(ACGIH)h
MAK (Germany)1
MAK Peak Exposure
(Germany)"
MAC (The
Netherlands)1"
OEL-LLV (Sweden)1
OEL-CLV (Sweden)"1
Exposure Duration
10 minute
0.50 ppm
20ppm
34 ppm
30 minute
0.50 ppm
15 ppm
25 ppm
20 ppm
1 ppm
1 ppm
5 ppm
5 ppm
5 ppm
1 hour
0.50 ppm
12 ppm
20 ppm
1 ppm
15 ppm
30 ppm
1 ppm
4 hour
0.50 ppm
8.2 ppm
14 ppm
0.25 ppm
8 hour
0.50 ppm
6.7 ppm
11 ppm
0.12 ppm
5 ppm (C)
3 ppm
5 ppm
2.0 ppm
2 ppm
1 a ERPG (Emergency Response Planning Guidelines, American Industrial Hygiene Association)
2 (AIHA 2003)
3 The ERPG-1 is the maximum airborne concentration below which it is believed nearly all individuals
4 could be exposed for up to one hour without experiencing other than mild, transient adverse health
5 effects or without perceiving a clearly defined objectionable odor.
6 The ERPG-2 is the maximum airborne concentration below which it is believed nearly all individuals
7 could be exposed for up to one hour without experiencing or developing irreversible or other serious
8 health effects or symptoms that could impair an individual's ability to take protective action.
9 The ERPG-3 is the maximum airborne concentration below which it is believed nearly all individuals
10 could be exposed for up to one hour without experiencing or developing life-threatening health effects.
11
12 bEEGL (Emergency Exposure Guidance Levels) National Research Council (NRC 1985) The EEGL is
13 the concentration of contaminants that can cause discomfort or other evidence of irritation or
14 intoxication in or around the workplace, but avoids death, other severe acute effects and long-term
15 or chronic injury.
16
17 CIDLH (Immediately Dangerous to Life and Health, National Institute of Occupational Safety and
18 Health) (NIOSH 1996) represents the maximum concentration from which one could escape
19 within 30 minutes without any escape-impairing symptoms, or any irreversible health effects.
20 The IDLH for nitrogen dioxide is based on acute inhalation toxicity data in humans.
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1
2 dNIOSH REL-STEL (Recommended Exposure Limits - Short Term Exposure Limit) (NIOSH
3 2003) is defined analogous to the ACGIH TLV-STEL.
4
5 eOSHA PEL-STEL (Permissible Exposure Limits - Short Term Exposure Limit) (NIOSH
6 2003) is defined analogous to the ACGIH-TLV-STEL.
7
8 fOSHA PEL-TWA (Occupational Health and Safety Administration, Permissible Exposure Limits -
9 Ceiling)
10 (OSHA 1999) is defined analogous to the ACGIH-TLV-TWA, but is for exposures of no
11 more than 10 hours/day, 40 hours/week. (C) denotes a ceiling.
12
13 gACGIH TLV-TWA (American Conference of Governmental Industrial Hygienists, Threshold
14 Limit Value - Time Weighted Average) (ACGIH 2003) is the time-weighted average
15 concentration for a normal 8-hour workday and a 40-hour workweek, to which nearly all workers
16 may be repeatedly exposed, day after day, without adverse effect.
17
18 hACGIH TLV-STEL (Threshold Limit Value - Short Term Exposure Limit) (ACGIH 2003) is
19 defined as a ISminute TWA exposure which should not be exceeded at any time during the
20 workday even if the 8-hour TWA is within the TLV-TWA. Exposures above the TLV-TWA up
21 to the STEL should not be longer than 15 minutes and should not occur more than 4 times per
22 day. There should be at least 60 minutes between successive exposures in this range.
23
24 'MAK (Maximale Argeitsplatzkonzentration [Maximum Workplace Concentration]) (Deutsche
25 Forschungsgemeinschaft [German Research Association] 2002) is defined analogous to the
26 ACGIH-TLV-TWA.
27
28 JMAK Spitzenbegrenzung (Peak Limit [Category 1,1]) (German Research Association 2002) constitutes
29 the average concentration to which workers can be exposed for a period up to 15 minutes with no
30 more than 1 excursion per work shift and a minimum of 1 hour between excursions.
31
32 kMAC (Maximaal Aanvaaarde Concentratie [Maximal Accepted Concentration]) (SDU
33 Uitgevers [under the auspices of the Ministry of Social Affairs and Employment], The
34 Hague, The Netherlands 2000) is defined analogous to the ACGIH-TLV-TWA.
35
36 'OEL-LLV (Occupational Exposure Limits - Level Limit Value) (Swedish National Board of
37 Occupational Safety and Health 1996) is an occupational exposure limit value for exposure during
38 one working day.
39
40 mOEL-CLV (Occupational Exposure Limits - Ceiling Limit Value) (Swedish National Board of
41 Occupational Safety and Health 1996) is an occupational exposure limit value for exposure
42 during a reference period of fifteen minutes.
43
44 8.3. Data Adequacy and Research Needs
45
46 Data on the effects of NC>2 on asthmatics and individuals with respiratory disease
47 were inconsistent and inconclusive. Additional studies that correlate severity of disease
48 with individual responses would be helpful.
49
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1 9. REFERENCES
2
3 Abe, M. 1967. Effects of mixed NO2-SO2 gas on human pulmonary functions. Bull. Tokyo Med. Dent.
4 Univ. 14:415-433.
5
6 Abraham, W.M., Welker, M., Oliver, W., Jr., Mingle, M., Januszkiewicz, A.J., Wanner, A., and Sackner,
7 M. 1980. Cardiopulmonary effects of short-term nitrogen dioxide exposure in conscious sheep.
8 Environ. Res. 22:61-72.
9
10 ACGIH. 1991. American Conference of Governmental Industrial Hygienists, Inc. Nitrogen Dioxide. In:
11 Documentation of the Threshold Limit Values and Biological Exposure Indicies, 6th ed., ACGIH,
12 Cincinnati, OH, pp. 11081110.
13
14 ACGIH. 2003. American Conference of Governmental Industrial Hygienists, Inc. TLVs and BEIs
15 Based on the Documentation of the Threshold Limit Values for Chemical Substances and
16 Physical Agents and Biological Exposure Indicies. ACGIH, Cincinnati, OH, pp. 44.
17
18 AIHA (American Industrial Hygiene Association). 2003. The AIHA 2003 Emergency Response Planning
19 Guidelines and Workplace Environmental Exposure Level Handbook. Amer. Ind. Hyg. Assoc.,
20 Fairfax, Virginia.
21
22 Adams, W.C., Brookes, K.A., and Schelegle, E.S. 1987. Effects of NO2 alone and in combination with O3
23 on young men and women. J. Appl. Physiol. 62:1698-1704.
24
25 Ainslie, G. 1993. Inhalational injuries produced by smoke and nitrogen dioxide. Respir. Med. 87:169-174.
26
27 Azoulay-Dupuis, E., Torres, M., Soler, P., and Moreau, J. 1983. Pulmonary NO2 toxicity in neonate
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2 Mostardi, R.A., Woebkenberg, N.R., Ely, D., Atwood, G., Conlon, M., Jarrett, M., and Dahlin, M. 1981.
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18
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22
23
24 NIOSH. 2003. National Institute for Occupational Safety and Health. NIOSH Pocket Guide to
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39
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42
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38 Robertson, A., Dodgson, J., Collings, P., and Seaton, A. 1984. Exposure to oxides of nitrogen: respiratory
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44
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48
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51
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55
56 Russell, M.L., Need, J.L., Mercer, R.R., Miller, F.J., and Crapo, J.D. 1991. Distribution of inhaled
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20
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24
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27
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36 26:422-437.
37
38 Shima, M. and Adachi, M. 2000. Effect of outdoor and indoor nitrogen dioxide on respiratory
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40
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43
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46
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21
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40
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42 Subpart H Prevention of Air Pollution Emergency Episodes. 40 CFR.
43
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47
48 U.S. EPA. 1990. U.S. Environmental Protection Agency. Health and Environmental Effects Document for
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50 133 pp.
51
52 U.S. EPA. 1993. U.S. Environmental Protection Agency. Air Quality Criteria for Oxides of Nitrogen, Vol.
53 I-III. Office of Research and Development, U.S. EPA, Research Triangle Park, NC.
54
55 U.S. EPA. 1995. U.S. Environmental Protection Agency. Review of the National Ambient Air Quality
56 Standards for Nitrogen Dioxide. Assessment of Scientific and Technical Information. OAQPS
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1 Staff Paper. Office of Air Quality, U.S. EPA, Research Triangle Park, NC. 95 pp.
2
3 U.S. EPA. 1997. U.S. Environmental Protection Agency. §50.11 National Primary and Secondary Ambient
4 Air Quality Standards for Nitrogen Dioxide, p. 8. 40 CFR.
5
6 Utell, M.J. 1989. Asthma and nitrogen dioxide: A review of the evidence. ASTM STP 1024, M.J. Utell
7 and R. Frank, Eds., American Society for Testing and Materials, Philadelphia, pp. 218-223.
8
9 Vagaggini, B., Paggiaro, P.L., Giannini, D., Di Franco, A., Cianchette, S., Carnevali, S., Taccola, M,
10 Bacci, E., Bancalari, L., Dente, F.L., and Giuntini, C. 1996. Effect of short-term NO2 exposure on
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12
13 Vollmuth, T.A., Driscoll, K.E., and Schlesinger, R.B. 1986. Changes in early alveolar particle
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15 Environ. Health 19:255-266.
16
17 von Nieding, G., Krekeler, H., and Fuchs, R. 1973. Studies of the acute effects of NO2 on lung
18 function: influence on diffusion, perfusion and ventilation in the lungs. Int. Arch. Arbeitsmed.
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20
21 von Nieding, G. and Wagner, H.M. 1979. Effects of NO2 on chronic bronchitics. Environ. Health Perspect.
22 29:137-142.
23
24 von Nieding, G., Wagner, H.M., Krekeler, H., Lollgen, H., Fries, W., and Beuthan, A. 1979. Controlled
25 studies of human exposure to single and combined action of NO2, O3, and SO2. Int. Arch. Occup.
26 Environ. Health 43:195-210.
27
28 Yue, M.-x., Peng, R.-y., Wang, Z.-g., Wang, D.-w., Yang, Z.-h., and Yang, H.m. 2004. Characteristics of
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30 Medical Eng. 17:117-120. (Chinese with English abstract; partially translated).
31
63
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
i APPENDIX A: Derivation of AEGL Values
2
64
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NITROGEN OXIDES
NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Key Study:
Toxicity endpoint:
Time scaling:
Uncertainty factors:
Modifying factor:
Calculations:
Derivation of AEGL-1 for Nitrogen Oxides
Kerretal., 1978, 1979
slight burning of the eyes, slight headache, chest tightness or
labored breathing with exercise in 7/13 asthmatics exposed to
0.5 ppm for 2 hours
Not applied
None
None
None; 0.50 ppm value applied across AEGL-1 exposure
durations
Appropriate chemical-specific data were not available for derivation of AEGL-1 values
for N2O4. NC>2 exists as an equilibrium mixture of NC>2 and ^64 but the dimer is not
important at ambient concentrations (U.S. EPA, 1993). When ^CMs released it
vaporizes and dissociates into NC>2. Thus, the AEGL values were developed based on
data for NO2 and are considered applicable to all nitrogen oxides. Values for N2O4 in
units of ppm have been calculated on a molar basis as presented in the text.
65
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NITROGEN OXIDES
NAC/Interim (NO2)/Proposed (N2O4): 12/2008
Derivation of AEGL-2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Key Studies:
Henschler et al., 1960
Toxicity endpoints: burning sensation in nose and chest, cough, dyspnea, sputum
production in normal volunteers exposed to 30 ppm for 2 hours
8 Time scaling:
C3'5 x t = k; the value of n was calculated by ten Berge et al. (1986)
from the data of Hine et al. (1970).
Uncertainty factors: 3 for intraspecies variability
Modifying factor: None
Appropriate chemical-specific data were not available for derivation of AEGL-2 values
for N2O4. NO2 exists as an equilibrium mixture of NO2 and N2O4 but the dimer is not
important at ambient concentrations (U.S. EPA, 1993). When N2C>4is released it
vaporizes and dissociates into NO2. Thus, the AEGL values were developed based on
data for NO2 and are considered applicable to all nitrogen oxides. Values for N2O4 in
units of ppm have been calculated on a molar basis as presented in the text.
Calculations:
C3'5 x t = k
(30 ppm/sy-3 x 2 hours = k
6324.56 ppm-hours = k
10-minute AEGL-2: C = (6324.56 ppm-hours/0.167 hours)
C = 20 ppm
30-minute AEGL-2: C = (6324.56 ppm-hours/0.5 hour)173'5
C = 15 ppm
1/3.5
1-hour AEGL-2:
4-hour AEGL-2:
8-hour AEGL-2:
C = (6324.56 ppm-hours/1 hour)173'5
C = 12 ppm
C = (6324.56 ppm-hours/4 hours)
C = 8.2 ppm
C = (6324.56 ppm-hours/8 hours)
C = 6.7 ppm
1/3.5
1/3.5
66
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1 Derivation of AEGL-3
2
3 Key Studies: Henry etal., 1969
4
5 Toxicity endpoint: signs of marked irritation, but no deaths in monkeys exposed to 50
6 ppm for 2 hours
7
8 Time scaling: C3'5 x t = k; the value of n was calculated by ten Berge et al. (1986)
9 from the data of Hine et al. (1970).
10
11 Uncertainty factors: 3 for intraspecies variability; 1 for interspecies variability
12
13 Modifying factor: None
14
15 Appropriate chemical-specific data were not available for derivation of AEGL-3 values
16 for N2O4. NO2 exists as an equilibrium mixture of NO2 and N2O4 but the dimer is not
17 important at ambient concentrations (U.S. EPA, 1993). As a result when N2O4 is released
18 it vaporizes and dissociates into NO2. Thus, the AEGL values were developed based on
19 data for NO2 and are considered applicable to all nitrogen oxides. Values for N2O4 in
20 units of ppm have been calculated on a molar basis as presented in the text.
21
22 Calculations: C3'5 x t = k
23 (50 ppm/3)3'5 x 2 hours = k
24 37,801 ppm-hours = k
25
26 10-minute AEGL-3: C = (37,801 ppm-hours/0.1667 hours)173'5
27 C = 34 ppm
28
29 30-minute AEGL-3: C = (37,801 ppm-hours/0.5 hours)173'5
30 C = 25 ppm
31
32 1-hour AEGL-3: C = (37,801 ppm-hours/1 hours)173'5
33 C = 20 ppm
34
35 4-hour AEGL-3: C = (37,801 ppm-hours/4 hours)173'5
36 C = 14 ppm
37
38 8-hour AEGL-3: C = (37,801 ppm-hours/8 hours)173'5
39 C = 11 ppm
40
67
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
i APPENDIX B: Derivation Summary for AEGL Values for Nitrogen
2 Oxides
68
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NITROGEN OXIDES
NAC/Interim (NO2)/Proposed (N2O4): 12/2008
AEGL-1 VALUES for Nitrogen Dioxide and Nitrogen Tetroxide
Chemical
NO2
N204
10 minute
0.94 mg/m3
(0.50 ppm)
0.94 mg/m3
(0.25 ppm)
30 minute
0.94 mg/m3
(0.50 ppm)
0.94 mg/m3
(0.25 ppm)
1 hour
0.94 mg/m3
(0.50 ppm)
0.94 mg/m3
(0.25 ppm)
4 hour
0.94 mg/m3
(0.50 ppm)
0.94 mg/m3
(0.25 ppm)
8 hour
0.94 mg/m3
(0.50 ppm)
0.94 mg/m3
(0.25 ppm)
Key Reference:
Keir, H.D., Kulle, T.J., Mcllhany, M.L., and Swidersky, P. 1978.
Effects of nitrogen dioxide on pulmonary function in human subjects. An
environmental chamber study. Report: ISS EPA/600/1-78/025; Order
no. PB-281 186,20pp.
Kerr, H.D., Kulle, T.J., Mcllhany, M.L., and Swidersky, P. 1979.
Effects of nitrogen dioxide on pulmonary function in human subjects:
An environmental chamber study. Environ. Research 19:392-404.
Test Species/Strain/Number: Human subjects; sex not given; 13 asthmatics with exercise
Exposure Route/Concentrations/Durations:
Inhalation: 0.5 ppm NO2 for 2 hours
Effects: slight burning of the eyes, slight headache, chest tightness, or labored
breathing in 7/13 subjects
Endpoint/Concentration/Rationale:
Mild symptoms of discomfort in asthmatics.
Uncertainty Factors/Rationale:
Total uncertainty factor: none
Interspecies: NA; human data used
Intraspecies: 1 - asthmatics were used as the test population
Modifying Factor: none
Animal to Human Dosimetric Adjustment: not applicable
Time Scaling: Extrapolation to time points was not conducted because adaptation to mild
sensory irritation occurs. In addition, animal responses to NO2 exposure have
demonstrated a much greater dependence upon concentration than upon time;
therefore, extending the 2-hour concentration to 8 hours should not exacerbate
the human response.
Data Quality and Support for the AEGL Values: AEGL-1 values are considered conservative
and should be protective of the toxic effects of NO2 outside those expected as defined under
AEGL-1.
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NITROGEN OXIDES
NAC/Interim (NO2)/Proposed (N2O4): 12/2008
AEGL-2 VALUES for Nitrogen Dioxide and Nitrogen Tetroxide
Chemical
NO2
N204
10 minute
38 mg/m3
(20 ppm)
38 mg/m3
(10 ppm)
30 minute
28 mg/m3
(15 ppm)
28 mg/m3
(7.6 ppm)
1 hour
23 mg/m3
(12 ppm)
23 mg/m3
(6.2 ppm)
4 hour
15 mg/m
(8.2 ppm)
15 mg/m
(4.1 ppm)
8 hour
13 mg/m
(6.7 ppm)
13 mg/m
(3.5 ppm)
Key Reference:
Henschler, D., Stier, A., Beck, H., and Neuman, W. 1960. Odor
threshold of a few important irritant gasses (sulfur dioxide, ozone,
nitrogen dioxide) and observations in humans exposed to low
concentrations. Archiv fur Gewerbepathologie und
Gewerbehygiene 17:547-570.
Test Species/Strain/Number: human, healthy male, 10-14
Exposure Route/Concentrations/Durations: 0.5-30 ppm for up to 2 hours
Effects:
0.5 ppm: metallic taste
1.5 ppm: dryness of the throat
4 ppm: sensation of constriction
25 ppm: prickling of the nose
30 ppm: burning sensation in nose and chest, cough, dyspnea, sputum production
Endpoint/C oncentrati on/Rati onal e:
Humans exposed to 30 ppm for 2 hours experienced
pronounced irritation. The point of departure is
considered a threshold for AEGL-2 since the effects
noted by the subjects would not impair the ability to
escape and the effects were reversible after cessation of
exposure.
Uncertainty Factors/Rationale:
Total uncertainty factor: 3
Interspecies: NA, human data used
Intraspecies: 3 - mechanism of action of a direct acting irritant is not expected
to differ greatly among individuals
Modifying Factor: not applicable
Animal to Human Dosimetric Adjustment: not applicable
Time Scaling: Cn x t = k where n = 3.5 (ten Berge et al., 1986)
Data Quality and Support for the AEGL Values: AEGL-2 values should be protective of the
toxic effects of NO2 outside those expected as defined under AEGL-2. The values are
supported by occupational monitoring data.
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NITROGEN OXIDES
NAC/Interim (NO2)/Proposed (N2O4): 12/2008
Chemical
NO2
N204
10 minute
64 mg/m
(34 ppm)
64 mg/m3
(17 ppm)
30 minute
47 mg/m3
(25 ppm)
47 mg/m3
(13 ppm)
1 hour
38 mg/m3
(20 ppm)
38 mg/m3
(10 ppm)
4 hour
26 mg/m3
(14 ppm)
26 mg/m3
(7.0 ppm)
8 hour
21 mg/m3
(11 ppm)
21 mg/m3
(5.7 ppm)
AEGL-3 VALUES for Nitrogen Dioxide and Nitrogen Tetroxide
Key Reference:
Henry, M.C., Ehrlich, R., and Blair, W.H. 1969. Effect of
nitrogen dioxide on resistance of squirrel monkeys to Klebsiella
pneumonias infection. Arch. Environ. Health 18:580-587.
Test Species/Strain/Number: monkeys, 2-6/group
Exposure Route/Concentrations/Durations: Inhalation, 10, 15, 35, 50 ppm for 2 hours
Effects:
50 ppm: marked increase in respiratory rate and decrease in tidal volume, microscopic
lesions in lung (determinate for AEGL-3)
35 ppm: increase in respiratory rate and decrease in tidal volume, microscopic lesions in
lung
10 and 15 ppm: slight changes in lung function
Endpoint/Concentration/Rationale: 50 ppm resulted in marked effects on lung function but
no deaths
Uncertainty Factors/Rationale:
Total uncertainty factor: 3
Interspecies:
Intraspecies:
1 - the endpoint in the monkey study is below the definition of
AEGL-3, human data support AEGL-3 point of departure and
derived values, the mechanism of action does not vary between
species with the target at the alveoli, and due to the similarities of
the respiratory tract between humans and monkeys
3 - mechanism of action of a direct acting irritant is not expected
to differ greatly among individuals.
Modifying Factor: not applicable
Animal to Human Dosimetric Adjustment: not applicable
Time Scaling: Cn x t = k where n = 3.5 (ten Berge et al., 1986)
Data Quality and Support for the AEGL Values: The study is of high quality and the AEGL-3
values are supported by human data.
71
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NITROGEN OXIDES NAC/Interim (NO2)/Proposed (N2O4): 12/2008
i APPENDIX C: Time Scaling Category Plot for Nitrogen Oxides
2
72
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NITROGEN OXIDES
NAC/Interim (NO2)/Proposed (N2O4): 12/2008
1000.0
Chemical Toxicity - TSD All Data
Nitrogen Dioxide
60 120 180 240 300 360 420 480
Minutes
Figure 1: Category plot of AEGL values and effects of nitrogen dioxide on humans and animals
Source
NAC/AEGL-
NAC/AEGL-
NAC/AEGL-
NAC/AEGL-
NAC/AEGL-
NAC/AEGL-2
NAC/AEGL-2
NAC/AEGL-2
NAC/AEGL-2
NAC/AEGL-2
NAC/AEGL-3
NAC/AEGL-3
NAC/AEGL-3
NAC/AEGL-3
NAC/AEGL-3
Norwood et al., 1966
Morley and Silk, 1970
Species
human
human
Sex
m
#
Exposures
ppm
0.5
0.5
0.5
0.5
0.5
20
15
12
8.2
6.7
34
25
20
14
11
90.0000
30.0000
Minutes
10
30
60
240
480
10
30
60
240
480
10
30
60
240
480
40
40
Category
AEGL
AEGL
AEGL
AEGL
AEGL
AEGL
AEGL
AEGL
AEGL
AEGL
AEGL
AEGL
AEGL
AEGL
AEGL
2
1
73
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NITROGEN OXIDES
NAC/Interim (NO2)/Proposed (N2O4): 12/2008
Henschleretal., 1960
multiple studies
Frampton et al, 1991
Linn and Hackney, 1983, 1984
von Nieding et al., 1979
Kleinman et al., 1983
Sackner et al., 1981
Kerretal., 1978
Roger etal., 1990
Roger etal., 1990
Mine et al., 1970
Mine et al., 1970
Mine et al., 1970
Mine etal., 1970
Henry etal., 1969
Mine et al., 1970
Carson et al., 1962
Carson et al., 1962
Carson et al., 1962
Mine et al., 1970
Henschler and Lutge, 1963
Bauer etal., 1985
Mine etal., 1970
Carson etal., 1962
Carson etal., 1962
Meulenbeltetal., 1992
Hidekazu and Fujio, 1981
Henschler and Lutke, 1963
Mine etal., 1970
Mine etal., 1970
human
human
human
human
human
human
human
human
human
human
dog
rat
mouse
rabbit
monkey
dog
rat
rat
rat
rat
human
human
guinea pig
rabbit
rat
rat
mouse
dog
guinea pig
mouse
30.0000
0.6000
1.5000
4.0000
5.0000
0.2000
1.0000
0.5
0.3000
0.6
75
100
100
75
50
20
190
90
72
20
20
0.3
50
315
115
200
40
40
20
20
120
180.0
180
75
120.0
120.0
240.0
120
110
75
240
240
240
60
120
1440
5
15
60
1440
120
240
60
15
60
10
720
360
1440
1440
1
0
0
0
1
0
0
1
1
0
PL
3
3
PL
2
1
2
2
2
1
1
1
PL
PL
PL
2
PL
0
1
1
For Category 0 = No effect, 1 = Discomfort, 2 = Disabling, PL = Partially Lethal, 3 = Lethal
74
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