Fax On Demand: Proposed 1: 1/ 2003 Telephone: ( 202) 401 0527 Item XXXX ACUTE EXPOSURE GUIDELINE LEVELS ( AEGLs)

FOR NITROGEN DIOXIDE ( CAS Reg. No. 10102 44 0)

PROPOSED NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 PREFACE 1 2 Under the authority of the Federal Advisory Committee Act ( FACA) P. L. 92 463 of 1972, 3 the National Advisory Committee for Acute Exposure Guideline Levels for Hazardous Substances 4 ( NAC/ AEGL Committee) has been established to identify, review and interpret relevant toxicologic 5 and other scientific data and develop AEGLs for high priority, acutely toxic chemicals. 6 7 AEGLs represent threshold exposure limits for the general public and are applicable to 8 emergency exposure periods ranging from 10 minutes to 8 hours. Three levels   AEGL 1, AEGL 2 9 and AEGL 3   are developed for each of five exposure periods ( 10 and 30 minutes, 1 hour, 4 hours, 10 and 8 hours) and are distinguished by varying degrees of severity of toxic effects. The three AEGLs 11 are defined as follows: 12 13 AEGL 1 is the airborne concentration ( expressed as parts per million or milligrams per cubic 14 meter [ ppm or mg/ m3]) of a substance above which it is predicted that the general population, 15 including susceptible individuals, could experience notable discomfort, irritation, or certain 16 asymptomatic, non sensory effects. However, the effects are not disabling and are transient and 17 reversible upon cessation of exposure. 18 19 AEGL 2 is the airborne concentration ( expressed as ppm or mg/ m3) of a substance above 20 which it is predicted that the general population, including susceptible individuals, could experience 21 irreversible or other serious, long lasting adverse health effects or an impaired ability to escape. 22 23 AEGL 3 is the airborne concentration ( expressed as ppm or mg/ m3) of a substance above 24 which it is predicted that the general population, including susceptible individuals, could experience 25 life threatening health effects or death. 26 27 Airborne concentrations below the AEGL 1 represent exposure levels that can produce mild 28 and progressively increasing but transient and nondisabling odor, taste, and sensory irritation, or 29 certain asymptomatic, non sensory effects. With increasing airborne concentrations above each 30 AEGL, there is a progressive increase in the likelihood of occurrence and the severity of effects 31 described for each corresponding AEGL. Although the AEGL values represent threshold levels for 32 the general public, including susceptible subpopulations, such as infants, children, the elderly, persons 33 with asthma, and those with other illnesses, it is recognized that individuals, subject to unique or 34 idiosyncratic responses, could experience the effects described at concentrations below the 35 corresponding AEGL. 36 37 38 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 2 TABLE OF CONTENTS 1 2 PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 4 LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5 6 LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 7 8 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 9 10 1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 11 12 2. HUMAN TOXICITY DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 13 2.1. Acute Lethality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 14 2.2. Nonlethal Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 15 2.2.1. Case Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 16 2.2.2. Epidemiologic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 17 2.2.3. Experimental Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 18 2.3. Developmental/ Reproductive Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 19 2.4. Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 20 2.5. Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 21 2.6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 22 23 3. ANIMAL TOXICITY DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 24 3.1. Acute Lethality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 25 3.1.1. Dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 26 3.1.2. Rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 27 3.1.3. Guinea Pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 28 3.1.4. Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 29 3.1.5. Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 30 3.2. Nonlethal Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 31 3.2.1. Monkeys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 32 3.2.2. Dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 33 3.2.3. Rabbits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 34 3.2.4. Sheep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 35 3.2.5. Guinea Pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 36 3.2.6. Hamsters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 37 3.2.7. Ferrets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 38 3.2.8. Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 39 3.2.9. Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 40 3.3. Developmental/ Reproductive Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 41 3.4. Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 3 3.5. Subchronic and Chronic Toxicity/ Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . 32 1 3.6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 2 3 4. SPECIAL CONSIDERATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4 4.1. Metabolism and Disposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 5 4.2. Mechanism of Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 6 4.3. Oxides of Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 7 4.4. Other Relevant Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 8 4.4.1. Species Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 9 4.4.2. Susceptible Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 10 4.4.3. Concentration Response Relationship . . . . . . . . . . . . . . . . . . . . . . . . . . 37 11 4.4.4. Susceptibility to infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 12 13 5. DATA ANALYSIS FOR AEGL 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 14 5.1. Summary of Human Data Relevant to AEGL 1 . . . . . . . . . . . . . . . . . . . . . . . . . . 38 15 5.2. Summary of Animal Data Relevant to AEGL 1 . . . . . . . . . . . . . . . . . . . . . . . . . . 39 16 5.3. Derivation of AEGL 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 17 18 6. DATA ANALYSIS FOR AEGL 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 19 6.1. Summary of Human Data Relevant to AEGL 2 . . . . . . . . . . . . . . . . . . . . . . . . . . 40 20 6.2. Summary of Animal Data Relevant to AEGL 2 . . . . . . . . . . . . . . . . . . . . . . . . . . 40 21 6.3. Derivation of AEGL 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 22 23 7. DATA ANALYSIS FOR AEGL 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 24 7.1. Summary of Human Data Relevant to AEGL 3 . . . . . . . . . . . . . . . . . . . . . . . . . . 42 25 7.2. Summary of Animal Data Relevant to AEGL 3 . . . . . . . . . . . . . . . . . . . . . . . . . . 42 26 7.3. Derivation of AEGL 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 27 28 8. SUMMARY OF AEGLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 29 8.1. AEGL Values and Toxicity Endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 30 8.2. Comparison with Other Standards and Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 31 8.3. Data Adequacy and Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 32 33 9. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 34 35 APPENDIX A: Derivation of AEGL Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 36 37 APPENDIX B: Derivation Summary for AEGL Values 38 for Nitrogen Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 39 40 APPENDIX C: Time Scaling Category Plot 41 for Nitrogen Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 4 LIST OF TABLES 1 2 Table 1. Physicochemical Data for Nitrogen Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3 Table 2. Effects of acute exposure to high NO 2 concentrations . . . . . . . . . . . . . . . . . . . . . . . . 11 4 Table 3. Summary of NO 2 mortality in five species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 5 Table 4. AEGL 1 values for Nitrogen Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 6 Table 5. AEGL 2 values for Nitrogen Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 7 Table 6. AEGL 3 values for Nitrogen Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 8 Table 7. Summary of AEGL values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 9 Table 8. Extant standards and guidelines for Nitrogen Dioxide . . . . . . . . . . . . . . . . . . . . . . . . 45 10 11 12 LIST OF FIGURES 13 14 15 Figure 1. Category plot of AEGL values and effects of nitrogen dioxide 16 on humans and animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 17 18 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 5 SUMMARY 1 2 NO 2 is an irritant to the mucous membranes and may cause coughing and dyspnea during 3 exposure. After less severe exposure, symptoms may persist for several hours before subsiding 4 ( NIOSH, 1976). With more severe exposure, pulmonary edema ensues with signs of chest pain, 5 cough, dyspnea, cyanosis, and moist rales heard on auscultation ( NIOSH, 1976; Douglas et al., 6 1989). Death from NO 2 inhalation is caused by bronchospasm and pulmonary edema in association 7 with hypoxemia and respiratory acidosis, metabolic acidosis, shift of the oxyhemoglobin dissociation 8 curve to the left, and arterial hypotension ( Douglas et al., 1989). A characteristic of NO 2 intoxication 9 after the acute phase is a period of apparent recovery followed by late onset bronchiolar injury that 10 manifests as bronchiolitis fibrosa obliterans ( NIOSH, 1976; NRC, 1977; Hamilton, 1983; Douglas 11 et al., 1989). In addition, experiments with laboratory animals indicate that exposure to NO 2 12 increases susceptibility to infection ( Henry et al., 1969; U. S. EPA, 1993). 13 14 For AEGL 1 a concentration of 0.5 ppm was adopted for all time points. Although the 15 response of asthmatics to NO 2 is variable, asthmatics were identified as a potentially susceptible 16 population. The evidence indicates that some asthmatics exposed to 0.3 0.5 ppm NO 2 may respond 17 with either subjective symptoms or slight changes in pulmonary function of no clinical significance. 18 In contrast, some asthmatics did not respond to NO 2 at concentrations of 0.5 4 ppm. Because of the 19 weight of evidence, the study by Kerr et al., ( 1978; 1979) was considered the most appropriate as the 20 basis of AEGL 1 values. They reported that 7/ 13 asthmatics experienced slight burning of the eyes, 21 slight headache, and chest tightness or labored breathing with exercise during exposure to 0.5 ppm 22 for 2 hours; at this concentration the odor of NO 2 was perceptible but the subjects became unaware 23 of it after about 15 minutes. No changes in any pulmonary function tests were found immediately 24 following the chamber exposure ( Kerr et al., 1978; 1979). Therefore, 0.5 ppm was considered a no 25 adverse effect level for the asthmatic population. Since asthmatics are potentially the most 26 susceptible population, no uncertainty factor was applied. 27 28 Supporting studies for AEGL 1 report findings similar to the key studies. Significant group 29 mean reductions in FEV 1 ( 17.3% with NO 2 vs  10.0% with air) and specific airway conductance ( 30 13.5% with NO 2 vs  8.5% with air) occurred in asthmatics after exercise during exposure to 0.3 ppm 31 for 4 hours and 1/ 6 individuals experienced chest tightness and wheezing ( Bauer et al., 1985). The 32 onset of effects was delayed when exposures were by oral nasal inhalation as compared to oral 33 inhalation and may result from scrubbing within the upper airway. In a similar study, asthmatics 34 exposed to 0.3 ppm for 30 minutes at rest followed by 10 minutes of exercise had significantly greater 35 reductions in FEV 1 ( 10% vs 4% with air) and partial expiratory flow rates at 60% of total lung 36 capacity, but no symptoms were reported ( Bauer et al., 1986). In a preliminary study with 13 37 asthmatics exposed to 0.3 ppm for 110 minutes, slight cough and dry mouth and throat and 38 significantly greater reduction ( 11% vs 7%) in FEV 1 occurred after exercise; however, in a larger 39 study, no changes in pulmonary function were measured and no symptoms were reported following 40 exposure of 21 asthmatics to concentrations up to 0.6 ppm for 75 minutes ( Roger et al., 1990). 41 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 6 Human data were also used as the basis for AEGL 2. Three healthy male volunteers 1 experienced definite discomfort from exposure to 30 ppm for 2 hours ( Henschler et al., 1960). Three 2 individuals exposed to 30 ppm for 2 hours perceived an intense odor upon entering the chamber, but 3 the odor perception quickly diminished and was completely absent after 25 40 minutes. One 4 individual experienced a slight tickling of the nose and throat mucous membranes after 30 minutes, 5 the two others after 40 minutes. From 70 minutes on, all subjects experienced a burning sensation 6 and an increasingly severe cough for the next 10 20 minutes, but coughing decreased from 100 7 minutes on. However, the burning sensation continued and moved into the lower sections of the 8 airways and was finally felt deep in the chest. At this time, marked sputum secretion and dyspnea was 9 noted. Towards the end of the exposure, the subjects' reported the exposure conditions to be 10 bothersome and barely tolerable. A sensation of pressure and increased sputum secretion continued 11 for several hours after cessation of exposure ( Henschler et al., 1960). 12 13 AEGL 3 values were based on animal data and supported by a human case report. Exposure 14 of monkeys to 50 ppm for 2 hours was used to derive the AEGL 3 values. Monkeys ( n = 2 6/ group) 15 were exposed to 10 50 ppm NO 2 for 2 hours with respiratory function monitored during exposure 16 ( Henry et al., 1969). NO 2 exposure alone resulted in a markedly increased respiratory rate and 17 decreased tidal volume during exposures to 50 and 35 ppm, but only slight effects at 15 and 10 ppm. 18 Mild histopathological changes in the lungs were noted after exposure to 10 and 15 ppm, however, 19 marked changes in lung structure were observed after exposure to 35 and 50 ppm. The alveoli were 20 expanded with septal wall thinning, bronchi were inflamed with proliferation or erosion of the surface 21 epithelium, and lymphocyte infiltration was seen with edema. In addition to the effects on the lungs, 22 interstitial fibrosis ( 35 ppm) and edema ( 50 ppm) of cardiac tissue, glomerular tuft swelling in the 23 kidney ( 35 and 50 ppm), lymphocyte infiltration in the kidney and liver ( 50 ppm), and congestion and 24 centrilobular necrosis in the liver ( 50 ppm) were observed. 25 26 The proposed AEGL 3 values are supported by human data for a welder. Pulmonary edema, 27 confirmed on X ray, resulted from exposure to approximately 90 ppm for up to 40 minutes 28 ( Norwood et al., 1966). If this exposure scenario is used for derivation of AEGL 3 values with an 29 uncertainty factory of 3 the values are nearly identical to those derived using the data in the monkey. 30 In addition, the proposed AEGL 3 values are below the concentrations at which lethality first 31 occurred in five animal species: 75 ppm for 4 hours in the dog and 1 hour in the rabbit, 50 ppm for 32 1 hour in the guinea pig, and 50 ppm for 24 hours in the rat and mouse ( Hine et al., 1970). In 33 general, the larger animals, including humans, are less susceptible to toxicity from NO 2 inhalation than 34 are the rodents. 35 36 For AEGL 2 and AEGL 3, the 10 and 30 minute, and 1, 4, and 8 hour AEGL endpoints 37 were calculated from Cn × t = k using n = 3.5 ( ten Berge et al., 1986). The value of n was calculated 38 by ten Berge et al. using the data of Hine et al. ( 1970) in five species of laboratory animal. An 39 uncertainty factor of 3 was applied to account for sensitive populations since the data for asthmatics 40 and those with respiratory disease are inconclusive. Because the endpoint in the monkey study is 41 below the definition of AEGL 3 and due to the similarities of the respiratory tract between humans 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 7 and monkeys, additional uncertainty factors are not considered necessary. Also, the mechanism of 1 action of NO 2 does not vary between species with the target at the alveoli. In general, the larger 2 animals, including humans, appear to be less susceptible to NO 2 than the rodents. 3 4 The proposed values for the three AEGL classifications for the four time periods are listed 5 in the table below. 6 7 8 Summary of AEGL Values for Nitrogen Dioxide 9 Classification 10 10 Minute 30 Minute 1 Hour 4 Hour 8 Hour Endpointa ( Reference)

AEGL   1b 11 ( Non 12 disabling) 13 0.50 ppm ( 0.94 mg/ m3) 0.50 ppm ( 0.94 mg/ m3) 0.50 ppm ( 0.94 mg/ m3) 0.50 ppm ( 0.94 mg/ m3) 0.50 ppm ( 0.94 mg/ m3) slight burning of the eyes, slight headache, chest tightness or labored breathing with exercies in 7/ 13 asthmatics ( Kerr et al., 1978, 1979)

AEGL   2 14 ( Disabling) 15 20 ppm ( 38 mg/ m3) 15 ppm ( 28 mg/ m3) 12 ppm ( 23 mg/ m3) 8.2 ppm ( 15 mg/ m3) 6.7 ppm ( 13 mg/ m3) burning sensation in nose and chest, cough, dyspnea, sputum production in normal volunteers ( Henschler et al., 1960)

AEGL   3 16 ( Lethal) 17 34 ppm ( 64 mg/ m3) 25 ppm ( 47 mg/ m3) 20 ppm ( 38 mg/ m3) 14 ppm ( 26 mg/ m3) 11 ppm ( 21 mg/ m3) marked irritation, no deaths, in monkeys ( Henry et al., 1969)

aSome effects may be delayed. 18 bThe sweet odor of NO 2 may be perceptible to most individuals at this concentration; however, adaptation occurs rapidly. 19 20 21 References 22 Bauer, M. A., Utell, M. J., Morrow, P. E., Speers, D. M., and Gibb, F. R. 1985. Route of inhalation 23 influences airway responses to 0.30 ppm nitrogen dioxide in asthmatic subjects. Am. Rev. 24 Respir. Dis. 131: A171. 25 26 Bauer, M. A., Utell, M. J., Morrow, P. E., Speers, D. M., and Gibb, F. R. 1986. Inhalation of 0.30 ppm 27 nitrogen dioxide potentiates exercise induced bronchospasm in asthmatics. Am. Rev. Respir. 28 Dis. 134: 1203 1208. 29 30 Douglas, W. W., Hepper, N. G. G., and Colby, T. V. 1989. Silo filler's disease. Mayo Clin. Proc. 31 64: 291 304. 32 33 Hamilton, A. 1983. Nitrogen compounds. In: Hamilton and Hardy's Industrial Toxicology, 4th ed. 34 Boston: John Wright PSG, Inc. pp. 184 186. 35 36 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 8 Henry, M. C., Ehrlich, R., and Blair, W. H. 1969. Effect of nitrogen dioxide on resistance of squirrel 1 monkeys to Klebsiella pneumoniae infection. Arch. Environ. Health 18: 580 587. 2 3 Henschler, D., Stier, A., Beck, H., and Neuman, W. 1960. Odor threshold of a few important 4 irritant gasses ( sulfur dioxide, ozone, nitrogen dioxide) and observations in humans exposed 5 to low concentrations. Archiv für Gewerbepathologie und Gewerbehygiene 17: 547 570. 6 ( translated from German) 7 8 Hine, C. H., Meyers, F. H., and Wright, R. W. 1970. Pulmonary changes in animals exposed to 9 nitrogen dioxide, effects of acute exposures. Toxicol. Appl. Pharmacol. 16: 201 213. 10 11 Kerr, H. D., Kulle, T. J., McIlhany, M. L., and Swidersky, P. 1978. Effects of nitrogen dioxide on 12 pulmonary function in human subjects. An environmental chamber study. Report: ISS 13 EPA/ 600/ 1 78/ 025; Order no. PB 281 186, 20 pp. 14 15 Kerr, H. D., Kulle, T. J., McIlhany, M. L., and Swidersky, P. 1979. Effects of nitrogen dioxide on 16 pulmonary function in human subjects: An environmental chamber study. Environ. Research 17 19: 392 404. 18 19 NIOSH. 1976. National Institute for Occupational Safety and Health. NIOSH criteria for a 20 recommended standard.... occupational exposure to oxides of nitrogen ( nitrogen dioxide and 21 nitric oxide). U. S. Department of Health, Education, and Welfare, Washington, D. C., HEW 22 publication No. ( NIOSH) 76 149, 195pp. 23 24 Norwood, W. D., Wisehart, D. E., Earl, C. A, Adley, F. E., and Anderson, D. E. 1966. Nitrogen 25 dioxide poisoning due to metal cutting with oxyacetylene torch. J. Occup. Med. 8: 301 306. 26 27 NRC. 1977. National Research Council. Medical and Biologic Effects of Environmental Pollutants. 28 Nitrogen Oxides. NRC, National Academy of Sciences, Washington, DC. 333pp. 29 30 Roger, L. J., Horstman, D. H., McDonnell, W., Kehrl, H., Ives, P. J., Seal, E., Chapman, R., and 31 Massaro, E. 1990. Pulmonary function, airway responsiveness, and respiratory symptoms 32 in asthmatics following exercise in NO 2. Toxicol. Ind. Health 6: 155 171. 33 34 ten Berge, W. F., Zwart, A., and Appelman, L. M. 1986. Concentration time mortality response 35 relationship of irritant and systemically acting vapours and gases. J. Hazard. Mat. 13: 301 36 309. 37 38 U. S. EPA. 1993. U. S. Environmental Protection Agency. Air Quality Criteria for Oxides of 39 Nitrogen, Vol. I III. Office of Research and Development, U. S. EPA, Research Triangle 40 Park, NC. 41 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 9 1. INTRODUCTION 1 2 Nitrogen dioxide ( NO 2) is the most ubiquitous of the oxides of nitrogen and has the greatest 3 impact on human health. NO 2, which exists as an equilibrium mixture of NO 2 and N 2 O 4 ( nitrogen 4 tetroxide), is a reddish brown gas with a sweet odor, is heavier than air, and reacts with water ( U. S. 5 EPA, 1993; Mohsenin, 1994). NO 2 is shipped under pressure and the equilibrium between NO 2 and 6 N 2 O 4 is altered with change in pressure with N 2 O 4 becoming predominant at very high pressures. 7 NO 2 is a free radical with sufficient stability to exist in relatively high concentrations in ambient air 8 ( Mohsenin, 1994). 9 10 The major source of atmospheric NO 2 is from the combustion of fossil fuels for heating, 11 household appliances, power generation, and in motor vehicles. Consequently, the chemical is a 12 major component of smog and a concern for indoor air quality. Ambient levels in urban air pollution 13 episodes in the US have been measured between 0.1 and 0.8 ppm as a maximum hourly average with 14 short term peaks as high as 1.27 ppm. Indoor NO 2 concentrations may reach a maximum 1 hour level 15 of 0.25 to 1.0 ppm with peak levels as high as 2 4 ppm where gas appliances or kerosene heaters are 16 used ( Mohsenin, 1994). 17 18 NO 2 is an irritant to the mucous membranes and may cause coughing and dyspnea during 19 exposure. After less severe exposure, symptoms may persist for several hours before subsiding 20 ( NIOSH, 1976). With more severe exposure, pulmonary edema ensues with signs of chest pain, 21 cough, dyspnea, cyanosis, and moist rales heard on auscultation ( NIOSH, 1976; Douglas et al., 22 1989). Death from NO 2 inhalation is caused by bronchospasm and pulmonary edema in association 23 with hypoxemia and respiratory acidosis, metabolic acidosis, shift of the oxyhemoglobin dissociation 24 curve to the left, and arterial hypotension ( Douglas et al., 1989). A characteristic of NO 2 intoxication 25 after the acute phase is a period of apparent recovery followed by late onset bronchiolar injury that 26 manifests as bronchiolitis fibrosa obliterans ( NIOSH, 1976; NRC, 1977; Hamilton, 1983; Douglas 27 et al., 1989). 28 29 Selected physicochemical properties of nitrogen dioxide are listed in Table 1. 30 31 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 10 TABLE 1. PHYSICOCHEMICAL DATA FOR NITROGEN DIOXIDE 12 Parameter 3 Value Reference Common name 4 nitrogen dioxide Synonyms 5 CAS registry no. 6 10102 44 0 Chemical formula 7 NO2 Budavari et al., 1996 Molecular weight 8 46.01 Budavari et al., 1996 Physical state 9 reddish brown gas Budavari et al., 1996 Vapor pressure 10 720 torr at 20

C; 800 mm Hg at 25

C ACGIH, 1991; U. S. EPA, 1990 Vapor density ( air = 1) 11 1.58 Budavari et al., 1996 Melting/ boiling point 12  9.3

C/ 21.15

C Budavari et al., 1996 Flamability 13 does not burn Budavari et al., 1996 Solubility in water 14 0.037 mL/ mL at 35

C Mohsenin, 1994 Conversion factors in air 15 1 ppm = 1.88 mg/ m3 1 mg/ m3 = 0.53 ppm U. S. EPA, 1993 Reactivity 16 decomposes in water forming nitric oxide and nitric acid Budavari et al., 1996 17 18 19 2. HUMAN TOXICITY DATA 20 2.1. Acute Lethality 21 22 Book ( 1982) used allometric scaling based on minute volumes and LC 50 values for NO 2 for 23 5 animal species to calculate a human 1 hour LC 50 of 174 ppm. Concentrations of > 200 ppm are 24 reported to induce immediate symptoms of bronchospasm and pulmonary edema and may cause 25 syncope, unconsciousness, and quick death ( Douglas et al., 1989). 26 27 Clinical responses to " acute" inhalation of high concentrations of NO 2, as summarized by 28 NRC ( 1977) and based on occupational exposures, are given in Table 2. Durations of exposures 29 were not specified except for the statement that workers in a nitric acid manufacturing plant in Italy 30 were exposed to average concentrations of 30 35 ppm for an unspecified number of years with no 31 adverse signs or symptoms. 32 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 11 1 Table 2: Effects of acute exposure to high NO2 concentrations 2 Concentration ( ppm) 3 Effect 0.4 4 approximate odor threshold 15 25 5 respiratory and nasal irritation 25 75 6 reversible pneumonia and bronchiolitis 150 300+ 7 fatal bronchiolitis and bronchopneumonia From NRC, 1977. 8 9 A man died after entering a well that had been blasted with gelignite, a nitro glycerine 10 explosive ( Derrick and Johnson, 1943). The individual appeared well for the next approximately 12 11 hours at which time he was taken to the hospital with dyspnea; he later fell into a coma and died. At 12 necropsy, the lungs appeared edematous with large areas of hemorrhage. About 29 hours after the 13 charge had been fired, concentrations of oxides of nitrogen in the well were less than 1 part in 14 100,000. 15 16 2.2. Nonlethal Toxicity 17 2.2.1. Case Reports 18 19 Probably the most well known manifestation of NO 2 toxicity is that of silo filler's disease. In 20 a silo, the gas that accumulates above the silage is depleted of oxygen, is rich in carbon dioxide, and 21 contains a mixture of nitrogen oxides, mainly NO 2 which can reach concentrations of 200 to 4000 22 ppm within 2 days ( Lowry and Schuman, 1956; Douglas et al., 1989). The term silo filler's disease 23 was first used by Lowry and Schuman in 1956 in an article in which they described the clinical 24 progression of the disease: inhalation of irritant gas from a silo; immediate cough and dyspnea with 25 a sensation of choking; 2 3 week period after exposure of apparent remission; second phase of illness 26 accompanied by fever with progressively more severe dyspnea, cyanosis, and cough; inspiratory and 27 expiratory rales; discrete nodular densities on the lung; and neutrophilic leukocytosis ( Lowry and 28 Schuman, 1956). Douglas et al. ( 1989) reported on 17 patients examined at the Mayo clinic between 29 1955 and 1987 after exposure to silo gas. Acute lung injury occurred in 11 individuals and 16 had 30 either persistent or delayed symptoms of dyspnea, cough, chest pain, eye irritation during exposure, 31 and rapid breathing. One patient died and at autopsy diffuse alveolar damage with hyaline membranes 32 and hemorrhagic pulmonary edema and acute edema of the airways were observed. Bronchiolitis 33 fibrosa obliterans developed in one patient many years later; however, prophylactic administration 34 of corticosteroids may have prevented chronic obstructive pulmonary disease ( COPD) in the other 35 patients. Similar case reports and outcomes of silo filler's disease and industrial exposures were 36 described in earlier literature ( Grayson, 1956; Lowry and Schuman, 1956; Milne, 1969). 37 38 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 12 A case was reported of a welder who developed shortness of breath and chest discomfort 1 during the use of an acetylene torch for metal cutting in a poorly ventilated water main; the worker 2 had spent approximately 30 minutes welding in the confined space before being forced to vacate. 3 Several hours later, the worker became so short of breath that he couldn't sleep that night. Chest X 4 ray 18 hours after exposure revealed pulmonary edema and a pulmonary function test showed 42% 5 of the predicted value for FVC. The individual was admitted to the hospital and treated with 6 antibiotics and oxygen. The patient fully recovered by 21 days after exposure. Simulation of the 7 accident produced an NO 2 concentration of 90 ppm within 40 minutes and total oxides of nitrogen 8 in excess of 300 ppm ( Norwood et al., 1966). It can be assumed that the individual was exposed to 9 at least 90 ppm during the welding operation and that the outcome could have been more severe, or 10 even fatal, without medical intervention. 11 12 An outbreak of NO 2 induced respiratory illness was reported among players and spectators 13 at two high school hockey games ( Hedberg et al., 1989). Patients presented with acute onset of 14 cough, hemoptysis, and/ or dyspnea during or within 48 hours of attending the hockey game. No 15 changes in lung function were measured 10 days and 2 months after exposure. NO 2 concentrations 16 were not measured in the arena during the outbreak, but the source was traced to a malfunctioning 17 motor on the ice resurfacer. Other cases of respiratory illness in hockey players, referees, and 18 spectators have been associated with elevated nitrogen dioxide levels in the arena due to 19 malfunctioning resurfacers or ventilation systems or combined with elevated carbon monoxide levels 20 ( Smith et al., 1992; Soparkar et al., 1993; Morgan, 1995; Karlson Stiber, et al., 1996). Attempts to 21 measure NO 2 concentrations in the arenas or reconstruct the situations were described by the authors 22 as not indicative of the actual exposure scenario which resulted in adverse effects. 23 24 Morley and Silk ( 1970) described a number of cases in which welders involved in ship repair 25 and shipbuilding were exposed to nitrous fumes. Symptoms included dyspnea, cough, headache, 26 tightness/ pain in chest, nausea, and cyanosis. Most patients recovered after treatment with oxygen 27 and antibiotics; however, one man died 43 days later from viral pneumonia. For two individuals 28 admitted to the hospital with cyanosis, dyspnea, and pulmonary edema, the concentration of NO 2 was 29 measured at 30 ppm during a 40 minute welding operation. However, the authors noted that 7 other 30 individuals present at the time were unaffected. 31 32 A railroad tank car ruptured at a chemical plant releasing a cloud of NO 2 in a small community 33 ( Bauer et al., 1998). In the first 30 hours after the release, the most common symptoms reported in 34 emergency room visits were headache, burning eyes, and sore throat. Most air samples collected 3 7 35 hours after the release showed concentrations of 0 ppm with one sample showing 1.4 ppm. No 36 attempt was made to correlate symptoms with estimated exposure. 37 38 Acute toxic reactions were described in four fireman who were exposed to NO 2 which 39 originated from a leak in a chemical plant ( Tse and Bockman, 1970). Concentrations were not 40 reported and exposure durations were defined as " barely a few minutes" to " about ten minutes". 41 Initial responses, which cleared within several days, included headache, a dry hacking cough, 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 13 pulmonary edema, sinusitis, and/ or upper respiratory tract irritation. Four to six weeks after 1 exposure, three of the patients developed fever, chest tightness, shortness of breath, and a productive 2 cough; these subsided and the patients remained asymptomatic. The fourth patient developed chronic 3 pulmonary insufficiency, consisting of dyspnea on exertion, despite normal chest X ray. 4 5 Four cases of exposure to unknown concentrations of nitrous fumes were reported for 6 individuals involved either in the use of an oxyacetylene burner, during a leak at a chemical plant, or 7 in shotfiring ( Jones et al., 1973). Three patients presented with pulmonary edema, one of which 8 progressed to bronchiolitis obliterans; the fourth patient presented with clinical features of 9 bronchiolitis obliterans. All recovered completely following corticosteroid treatment. 10 11 2.2.2. Epidemiologic Studies 12 13 Several epidemiological studies associating ambient NO 2 exposure with an increase in the 14 prevalence of respiratory illness have been inconclusive. Increased odds ratios ( 1.2 1.7) were found 15 for bronchitis, chronic cough, and chest illness but not for wheeze and asthma in children from six 16 U. S. cities with annual average NO 2 levels 0.0065 0.0226 ppm ( Dockery et al, 1989). No association 17 was found between long term differences in NO 2 levels ( change of 0.0106 ppm/ 6 week average) and 18 mean annual rates of respiratory episodes in children from urban and rural regions in Switzerland, 19 however the duration of symptoms was increased ( Braun Fahrlaender et al., 1992). An increase in 20 the cases of croup in children was associated with total suspended particulate matter and NO 2 21 ( Schwartz et al., 1991) and decreased lung function in children was linked to sulfur dioxide in 22 combination with NO 2 ( Mostardi et al., 1981). Symptoms of chronic obstructive pulmonary disease 23 have been linked to exposure to total oxidants (> 0.1 ppm), NO 2, and/ or sulfates, but not to NO 2 alone 24 ( Detels et al., 1981; Euler et al., 1988). Combined effects of NO 2, SO 2, particulate matter, H 2 S, and 25 other pollutants were considered as contributing factors to a positive association between the 26 occurrence of upper respiratory infections and living in polluted areas of Finland for children < 2 years 27 and 6 years old ( Jaakkola et al., 1991). 28 29 In a more recent study, children from 12 communities in California were assessed for 30 respiratory disease prevalence and pulmonary function ( Peters et al., 1999a, b). Wheeze prevalence 31 was correlated with levels of both acid and NO 2 in boys, whereas regression analysis showed that NO 2 32 was significantly associated with lower FVC, FEV 1, and maximal midexpiratory flow in girls. Similar 33 results were reported for eight areas of Switzerland in which a 10 µ g/ m3 average increase in NO 2 34 exposure was associated with decreases in FVC ( Schindler et al., 1998). 35 36 Epidemiological studies associating indoor NO 2 have also been inconclusive. One study found 37 no evidence for any short term association between prevalence of respiratory symptoms in infants and 38 median indoor and outdoor NO 2 levels of 6.8 and 12.6 ppb, respectively ( Farrow et al., 1997). Other 39 studies found a significant increase in the occurrence of sore throat, colds, and absences from school 40 among children exposed to hourly peak levels of

80 ppb NO 2 from unvented gas heating in the 41 classrooms ( Pilotto et al., 1997), increased respiratory illness in children from homes using gas 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 14 cooking where NO 2 concentrations in the children's bedroom ranged from 4 169 ppb ( Florey et al., 1 1979), and slight decreases in forced vital capacity and peak expiratory flow among adult asthmatics 2 exposed to > 0.3 ppm while cooking on a gas range ( Goldstein et al., 1988). Similar to these findings, 3 Neas et al. ( 1991) found that a 15 ppb increase in household annual NO 2 mean was associated with 4 an increased cumulative incidence of attacks of shortness of breath with wheeze, chronic wheeze, 5 chronic cough, chronic phlegm, or bronchitis in children. 6 7 As part of a review of the National Ambient Air Quality Standards ( NAAQS) for NO 2, U. S. 8 EPA ( 1995) conducted a meta analysis of studies which examined the respiratory effects on children 9 living in homes with gas stoves. Conclusions drawn from that analysis were that children ages 5 12 10 years old had an increased risk of about 20% for developing respiratory symptoms and disease with 11 each increase of 0.015 ppm in estimated 2 week average NO 2 exposure ( mean weekly concentrations 12 in bedrooms 0.008 0.065 ppm) and that no evidence for increased risk was found for infants < 2 years 13 old. Several limitations of this analysis were acknowledged including the uncertainty between 14 monitored vs. actual exposure concentration, that the method could not distinguish between peak and 15 average exposures, the confounding effects of other gas combustion by products, and in context of 16 the NAAQS review that indoor exposures do not mimic outdoor exposures ( U. S. EPA, 1995). 17 18 Several occupations result in exposure to NO 2 concentrations higher than ambient. In diesel 19 bus garage workers, NO 2 concentrations of

0.3 ppm, along with respirable particulates, were 20 associated with work related symptoms of cough, itching, burning or watering eyes, difficult 21 breathing, chest tightness, and wheeze; but, there were no reductions in pulmonary function ( Gamble 22 et al., 1987). In contrast, no relationship was found between exposure to oxides of nitrogen and 23 respiratory symptoms or decline in FEV 1 among British coalminers exposed to peak NO 2 24 concentrations up to 14 ppm; controls were matched for age, dust exposure, smoking habit, coal 25 rank, and type of work ( Robertson et al., 1984). No differences in pulmonary function were noted 26 among shipyard welders exposed to average concentrations of oxides of nitrogen of 0.04 ppm ( Peters 27 et al., 1973). Slight increases in prevalence of bronchitis ( 17.2% vs 12.6%) and colds ( 37.5% vs 28 30.7%) were noted in traffic officers exposed to automobile exhaust containing mean concentrations 29 of 0.045 0.06 ppm NO 2 ( Speizer and Ferris, 1973). 30 31 In conclusion, indoor air quality may be more significant than ambient air quality to the 32 prevalence of respiratory illness due to NO 2. A review of epidemiology studies which assessed 33 ambient quality ( U. S. EPA, 1993) yielded that there is insufficient evidence to make any conclusion 34 about the long or short term health effects of NO 2. Further, review of epidemiology studies that 35 assessed indoor air quality, found that meta analysis results yielded insufficient evidence to conclude 36 that NO 2 had an effect on infants 2 years and younger while several considerations limited the 37 interpretation of the positive results for children aged 5 12 years. 38 39 2.2.3. Experimental Studies 40 41 Healthy Subjects 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 15 The odor threshold for NO 2 in air has been reported as 0.4 ppm for recognition and 4.0 ppm 1 for < 100% identification ( NIOSH, 1976). In an experimental study, the odor of NO 2 was perceived 2 by 3/ 9 volunteers exposed to 0.12 ppm and by 8/ 13 subjects at 0.22 ppm. At concentrations of  4 3 ppm, the volunteers perceived the odor for 1 10 minutes, but the duration of perception was not 4 directly related to concentration. The olfactory response to NO 2 returned 1 1.5 minutes after 5 cessation of exposure ( Henschler et al., 1960). 6 7 Low level exposures of healthy individuals to NO 2 have shown no effects on pulmonary 8 function or symptoms. In several studies, healthy men and/ or women were exposed to 0.6 ppm NO 2 9 for 1 3 hours with intermittent or continuous exercise. No significant effects were observed in any 10 study on pulmonary function, cardiovascular function, metabolism, or symptoms of exposure 11 ( Folinsbee et al., 1978; Adams et al., 1987; Frampton et al., 1991; Hazucha et al., 1994). No changes 12 in pulmonary function occurred following exposure to 1.5 ppm for 3 hours or to a baseline of 0.05 13 ppm with intermittent peaks of 2 ppm, however, continuous exposure to 1.5 ppm for 3 hours resulted 14 in a slight but significantly greater fall in FEV 1 and FVC in response to carbachol ( Frampton et al., 15 1991). Pulmonary function was not affected in competitive athletes exposed to 0.18 and 0.30 ppm 16 for 30 minutes during heavy exercise ( Kim et al., 1991) or in healthy adults exposed to 0.3 ppm for 17 4 hours with intermittent exercise ( Smeglin et al., 1985). 18 19 Studies at higher concentrations of NO 2 indicate an apparent threshold before pulmonary 20 function is affected. No changes in pulmonary function, airway reactivity, or indications of irritation 21 were measured in healthy adults exposed to 1 ppm for 2 hours, 2 ppm for 3 hours ( Hackney et al., 22 1978), 2 ppm for 4 hours ( Devlin et al., 1992), 3 ppm for 2 hours ( Goings et al., 1989) or to 2.3 ppm 23 for 5 hours ( Rasmussen et al., 1992). Normal subjects exposed to 2 ppm for 1 hour developed an 24 increase in airway reactivity to methacholine challenge without changes in lung volume or pulmonary 25 function ( Mohsenin, 1988). No statistically significant effects on airway resistance, symptoms, heart 26 rate, skin conductance, or self reported emotional state were found in healthy volunteers exposed to 27 4 ppm NO 2 for 1 hour and 15 minutes with intermittent light and heavy exercise ( Linn and Hackney, 28 1983). However, a significant decrease in mean ( n = 11) alveolar O 2 partial pressure by 8 mm Hg 29 and a significant increase in mean ( n = 11) airway resistance from 1.51 to 2.41 cm H 2 O/( L/ s) occurred 30 in healthy volunteers exposed to 5 ppm for 2 hours with 6/ 11 individuals responding ( von Nieding 31 et al., 1979). 32 33 Henschler et al. ( 1960) performed several experiments on healthy, male volunteers. They 34 reported that a 2 hour exposure to 20 ppm did not cause any irritation as long as several exposures 35 to lower concentrations occurred during the preceding days; however, exposure to 30 ppm for 2 36 hours caused definite discomfort. Three individuals exposed to 30 ppm for 2 hours perceived an 37 intense odor upon entering the chamber which quickly diminished and was completely absent after 38 25 40 minutes. One individual experienced a slight tickling of the nose and throat mucous 39 membranes after 30 minutes, the two others after 40 minutes. From 70 minutes on, all subjects 40 experienced a burning sensation and an increasingly severe cough for the next 10 20 minutes, but 41 coughing decreased from 100 minutes on. However, the burning sensation continued and moved into 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 16 the lower sections of the airways and was finally felt deep in the chest. At this time, marked sputum 1 secretion and dyspnea was noted. Towards the end of the exposure, the subjects' reported the 2 exposure conditions to be bothersome and barely tolerable. A sensation of pressure and increased 3 sputum secretion continued for several hours after cessation of exposure ( Henschler et al., 1960). 4 5 In a similar experiment ( Henschler and Lütke, 1963) groups of 4 or 8 healthy, male volunteers 6 were exposed to 10 ppm for 6 hours or to 20 ppm for 2 hours. All subjects upon entering the 7 chamber noted the odor which diminished rapidly. At 20 ppm minor scratchiness of the throat was 8 felt after about 50 minutes and 3/ 8 experienced slight headaches towards the end of the exposure 9 period. Methemoglobin levels remained within the normal range in all subjects after exposure. 10 11 Biochemical changes in bronchoalveolar lavage fluid ( BALF) and blood have also been 12 studied following exposure of healthy adults to NO 2. Exposures to 2 ppm for 4 ( Devlin et al., 1992) 13 or 6 hours ( Frampton et al., 1992) caused an influx of polymorphonuclear leukocytes in BALF, 2.3 14 ppm for 5 hours resulted in a decrease in serum glutathione peroxidase activity ( Rasmusen et al., 15 1992), 1 and 2 ppm for 3 hours caused a decrease in RBC membrane acetylcholinesterase activity, 16 2 ppm for 3 hours resulted in an increase in peroxidized RBC lipids and glucose 6 phosphate 17 dehydrogenase activity ( Posin et al., 1978), and exposure to 3 or 4 ppm for 3 hours resulted in a 18 decrease in   1 protease inhibitor activity but not in overall enzyme levels in BALF ( Mohsenin and 19 Gee, 1987). Following exposure to 2 ppm for 4 hours, neutrophilic inflammation was detected in 20 bronchial washings but no changes in inflammatory cells were observed in endobronchial biopsy 21 samples ( Blomberg et al., 1997). Mucociliary activity was completely stopped in healthy individuals 22 45 minutes after exposure to 1.5 and 3.5 ppm for 20 minutes ( Helleday et al., 1995). 23 24 Asthmatic Subjects 25 26 Studies on the effects of NO 2 on pulmonary function in asthmatics are inconclusive and 27 conflicting. No consistent changes in pulmonary function or reported symptoms have been found in 28 exercising asthmatic adults and adolescents exposed to 0.12 or 0.18 ppm for 40 minutes ( Koenig et 29 al., 1987), 0.12 ppm for 1 hour at rest ( Koenig et al., 1985), 0.2 ppm for 2 hours with intermittent 30 exercise ( Kleinman et al., 1983), 0.3 ppm for 30 minutes ( Rubinstein et al., 1990), 1 hour ( Vagaggini 31 et al., 1996), or 4 hours with exercise ( Morrow and Utell, 1989), 0.5 ppm for 1 hour at rest 32 ( Mohsenin, 1987), up to 0.6 ppm for 75 minutes with intermittent exercise ( Roger et al., 1990), and 33 up to 1 ppm for 4 hours ( Sackner et al., 1981). No statistically significant differences between 34 control and NO 2 exposure were found for airway resistance, symptoms, heart rate, skin conductance, 35 or self reported emotional state for asthmatics exposed to 4 ppm for 75 minutes with intermittent 36 exercise ( Linn and Hackney, 1984). 37 38 Kerr et al. ( 1978, 1979) studied of the effects of NO 2 on pulmonary function as well as 39 reported symptoms which were not included in many other studies. The subjects were asked to keep 40 note of symptoms they experienced during exposure to 0.5 ppm for 2 hours, specifically cough, 41 sputum, irritation of mucus membranes, and chest discomfort. At this concentration the odor of NO 2 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 17 was perceptible but the subjects became unaware of it after about 15 minutes. Seven of 13 asthmatics 1 reported symptoms with exposure compared with only one of the 10 normal subject and one of the 2 7 subjects with chronic bronchitis. In the group of asthmatics, two had slight burning of the eyes, one 3 had a slight headache, three reported chest tightness, and one had labored breathing with exercise 4 compared with slight nasal discharge in the normal and chronic bronchitis individuals. No changes 5 in any pulmonary function tests were found immediately following the chamber exposure. 6 7 Significant group mean reductions in FEV 1 ( 17.3% with NO 2 vs  10.0% with air) and specific 8 airway conductance ( 13.5% with NO 2 vs  8.5% with air) occurred in asthmatics after exercise during 9 exposure to 0.3 ppm for 4 hours and 1/ 6 individuals experienced chest tightness and wheezing ( Bauer 10 et al., 1985). The onset of effects was delayed when exposures were by oral nasal inhalation 11 compared to oral inhalation and may result from scrubbing within the upper airway. In a similar 12 study, asthmatics exposed to 0.3 ppm for 30 minutes at rest followed by 10 minutes of exercise had 13 significantly greater reductions in FEV 1 ( 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 preliminary study 15 with 13 asthmatics exposed to 0.3 ppm for 110 minutes, slight cough and dry mouth and throat and 16 significantly greater reduction ( 11% vs 7%) in FEV 1 occurred after exercise, however, in a larger 17 study, no changes in pulmonary function were measured and no symptoms were reported following 18 exposure of 21 asthmatics to concentrations up to 0.6 ppm for 75 minutes ( Roger et al., 1990). The 19 mean drop in FEV 1 for asthmatics during a 3 hour exposure with intermittent exercise to 1 ppm NO 2 20 ( 2.5%) was significantly greater than the drop during air ( 1.3%) exposure; in BALF, levels of 6 keto 21 prostaglandin 1  were decreased and levels of thromboxane B 2 and prostaglandin D 2 were increased 22 after NO 2 exposure ( Jörres et al., 1995). 23 24 The effects of NO 2 on airway hyperreactivity in asthmatics have also been inconclusive among 25 studies. Methacholine responsiveness in asthmatics was not increased following exposure to 0.25 26 ppm for 20 minutes at rest plus 10 minutes of exercise ( Jörres and Magnussen, 1991) or by exposure 27 to 0.1 ppm for 1 hour at rest ( Hazucha et al., 1983). Exposure to 0.1 ppm for 1 hour caused a slight 28 but significant increase in initial specific airway resistance and enhanced the bronchoconstrictor effect 29 of carbachol in 13/ 20 asthmatics, but the remaining 7 subjects were unaffected. When the study was 30 repeated in 4 individuals ( two responders and two non responders) at 0.2 ppm, the results were 31 variable in that the two non responders were still unaffected, while one responder had an equal 32 response and the other had a greater response to carbachol challenge compared with their responses 33 to 0.1 ppm ( Orehek et al., 1976). Slight but significant potentiation of airway reactivity in asthmatics 34 occurred from NO 2 exposures of 0.5 ppm for 1 hour followed by methacholine challenge ( Mohsenin, 35 1987), 0.3 ppm for 40 minutes followed by isocapnic cold air hyperventilation ( Bauer et al., 1986), 36 0.2 ppm for 2 hours followed by methacholine challenge ( Kleinman et al., 1983), and 0.25 ppm for 37 30 minutes followed by isocapnic hyperventilation ( Jörres and Magnussen, 1990). A significantly 38 greater fall in FEV 1 from challenge with house dust mite antigen was reported for asthmatics as 39 compared to controls ( 7.76% vs  2.85%) following exposure to 0.4 ppm for 1 hour ( Tunnicliffe et 40 al., 1994), but no significant changes were found in a similar study using a 6 hour exposure ( Devalia 41 et al., 1994). Exposure of asthmatics to 0.4 ppm for 3 hours significantly decreased the amount of 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 18 inhaled allergen required to decrease FEV 1 by 20% but no changes in airway responsiveness occurred 1 following exposure to 0.2 ppm for 6 hours; these results suggest a concentration threshold rather than 2 a duration effect ( Jenkins et al., 1999). 3 4 Folinsbee ( 1992) conducted a meta analysis of twenty studies which measured airway 5 responsiveness in asthmatics following NO 2 exposure. Eight different agents were used in these 6 studies to induce non specific airway responsiveness and the analysis was limited to exposures in the 7 range of 0.2 to 0.3 ppm. For asthmatics, the fraction of subjects with an increase in airway 8 responsiveness was significant ( p  0.01) following exposures at rest, but not with exercise. When 9 only those studies which used a cholinergic agonist were analyzed similar results were found in that 10 a greater proportion of subjects showed an increased response in resting exposures than in exercising 11 exposures. 12 13 Subjects with Chronic Lung Disease 14 15 The results of studies on the effects of NO 2 on pulmonary function in patients with chronic 16 lung disease or bronchitis are also conflicting. No significant differences in pulmonary function or 17 symptom reporting were observed in patients with chronic respiratory illness exposed to 0.3 ppm for 18 4 hours at rest ( Hackney et al., 1992), in patients with chronic obstructive pulmonary disease ( COPD) 19 exposed to NO 2 levels up to 2 ppm for 1 hour with intermittent exercise ( Linn et al., 1985), and in 20 patients with chronic bronchitis exposed to 0.5 ppm for 2 hours with exercise ( Kerr et al., 1978, 21 1979). In contrast to these reports, forced expiratory volume of COPD patients significantly 22 decreased from 18.8 L after air to 13.6 L after exposure to 0.3 ppm for 1 hour ( Vagaggini et al., 23 1996). A significant reduction in forced vital capacity that progressed during exercise ( 1.2 to  8.2%) 24 occurred in elderly COPD patients exposed to 0.3 ppm for 4 hours while no effects were seen in an 25 age and gender matched healthy control group ( Morrow and Utell, 1989; Morrow et al., 1992). 26 27 The effects of NO 2 on respiratory gas exchange was investigated in patients with chronic 28 bronchitis. Inhalation of 4 and 5 ppm for 15 60 minutes significantly decreased the CO diffusing 29 capacity and arterial pO 2 with no progressive changes noted over time. Exposure to 5 ppm over 15 30 minutes resulted in an average decrease in CO diffusion capacity of 3.8 mL/ min/ torr and a decrease 31 in arterial pO 2 from an average of 76.5 to 71.3 torr. A slight, but statistically significant, increase in 32 airway resistance ( approximately 20 30% above the initial value) was measured for exposure 33 concentrations of 1.6 5 ppm for 5 minutes; no effects occurred at or below 1.5 ppm ( von Nieding et 34 al., 1973; von Nieding and Wagner, 1979). 35 36 2.3. Developmental/ Reproductive Toxicity 37 38 No information was found regarding the developmental or reproductive toxicity of NO 2 in 39 humans. 40 41 2.4. Genotoxicity 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 19 No information was found regarding the genotoxicity toxicity of NO 2 in humans. 1 2 2.5. Carcinogenicity 3 4 No information was found regarding the carcinogenicity of NO 2 in humans. 5 6 2.6. Summary 7 8 In humans, NO 2 exposure causes immediate irritation with pulmonary edema followed by a 9 latent period of apparent recovery. A second phase of symptoms can occur after several hours with 10 the development of bronchiolitis obliterans. An estimation of the concentration causing death in 11 humans is approximately

150 ppm, but no duration of exposure was given. Most case reports do 12 not contain concentrations or durations of exposure, however, welders exposed to 30 and 90 ppm 13 for 40 minutes experienced varying degrees of dyspnea, cough, headache, chest tightness, nausea, and 14 cyanosis with hospitalization required for pulmonary edema, confirmed by X ray, at the higher 15 concentration ( Norwood et al., 1966; Morley and Silk, 1970). 16 17 Epidemiological studies on the long term effects of elevated NO 2 have been conflicting. It 18 is likely that increases in respiratory illnesses are due to NO 2 in combination with other pollutants and 19 that short term peak concentrations are more detrimental than chronic, low level exposures. 20 Evidence suggests that children ages 5 12 have a greater risk for developing respiratory disease from 21 long term exposure to higher concentrations, but that infants do not. 22 23 Experimental studies with both healthy and asthmatic individuals are inconclusive. Negative 24 results have been obtained in many studies with exposures up to 4 ppm for 1 hour, however, other 25 studies report positive effects on pulmonary function at lower concentrations. It should be noted that 26 in the studies which found statistically significant changes with NO 2 exposure, the differences were 27 < 10% and of questionable biological significance even for asthmatics. However, the available 28 evidence also suggests that asthmatics may experience an increase in airway responsiveness from 29 concentrations of 0.2 0.3 ppm. 30 31 32 3. ANIMAL TOXICITY DATA 33 3.1. Acute Lethality 34 35 Acute lethality data were located for several species. One group of investigators ( Hine et al., 36 1970) studied the effects of varying concentration and duration of exposure in five different species 37 of laboratory animal; these results are described separately by species below and summarized in Table 38 3 at the end of this section. In this study, deaths generally occurred within 2 8 hours after exposure 39 and the majority within 24 hours. Additional data from rabbit, rat, and mouse studies were available 40 and are in good agreement with the results of the Hine et al. study. 41 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 20 3.1.1. Dogs 1 2 Greenbaum et al. ( 1967) exposed mongrel dogs ( n = 1/ exposure) to 0.1% ( 1000 ppm) NO 2 3 for 136 minutes, 0.5% ( 5000 ppm) for 5 45 minutes, or to 2% ( 20,000 ppm) for 15 minutes. All 4 dogs died that were exposed to either 0.5% for 35 or 45 minutes or to 2% for 15 minutes. 5 Respiration became shallow and gasping with deaths due to pulmonary edema. Fluid was visible in 6 the tracheobronchial tree at necropsy. Cyanosis due to methemoglobin formation ( 78%) was noted 7 in one animal given 2% for 15 minutes. At concentrations of 0.5% and 2%, arterial pO 2 and systemic 8 arterial pressure were reduced. The authors stated that pulmonary edema was caused by the action 9 of nitrogen dioxide on the alveolar lining fluid, forming nitric and nitrous acids which, in turn, cause 10 denaturing of proteins, rupture of lysosomes, and the development of chemical pneumonitis. 11 12 Hine et al. ( 1970) studied the effects of varying concentration and duration of NO 2 exposure 13 on mongrel dogs. Animals ( n = 1 4) were exposed to 5 250 ppm NO 2 for periods of 30 minutes to 14 24 hours. At concentrations of

40 ppm, signs of toxicity included lacrimation, reddening of the 15 conjunctivae, and increased respiration which became labored and difficult as the concentration 16 increased. Mortalities were first observed at 75 ppm for 4 hours ( Table 3). Terminally, respiration 17 became gasping and spasmodic and lung edema was observed at necropsy. Histological observations 18 of the lungs following deaths were characterized by bronchiolitis, desquamated bronchial epithelium, 19 infiltration by polymorphonuclear cells, and edema. 20 21 3.1.2. Rabbits 22 23 The 15 minute LC 50 for the rabbit ( strain not specified; n = 5) was 315 ppm. Clinical signs 24 of toxicity included severe respiratory distress, eye irritation, 10 15% body weight suppression for 25 two days, and death; time to death varied from 30 minutes to 3 days. Gross pathology revealed 26 darkened areas about the surface of the lungs. Histopathology of the lungs of the survivors 7 and 21 27 days after exposure showed lesions consisting of focal accumulation of intraalveolar macrophages, 28 some proliferation of the alveolar lining epithelium, and varying amounts of inflammatory cells 29 ( Carson et al., 1962). 30 31 Hine et al. ( 1970) studied the effects of varying concentration and duration of NO 2 exposure 32 on rabbits ( strain not specified). Animals ( n = 2 8) were exposed to 5 200 ppm NO 2 for 30 minutes 33 up to 24 hours. At concentrations of

40 ppm, signs of toxicity included lacrimation, reddening of 34 the conjunctivae, and increased respiration which became labored and difficult as the intoxication 35 increased. Mortalities were first observed at 75 ppm for 60 minutes ( Table 3). Terminally, 36 respiration became gasping and spasmodic and lung edema was observed at necropsy. Histological 37 observations of the lungs following deaths were characterized by bronchiolitis, desquamated bronchial 38 epithelium, infiltration by polymorphonuclear cells, and edema. 39 40 In a similar study, rabbits ( strain not specified; n = 3) were exposed to 125, 175, 250, 400, 41 600, or 800 ppm NO 2 for 10 minutes ( Meulenbelt et al., 1994). Two of three animals given 800 ppm 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 21 died 7 21 hours after exposure. Lung weights were significantly higher and lung homogenates 1 contained greater amounts of protein, lactate dehydrogenase ( LDH), glutathione peroxidase, and 2 glucose 6 phosphate dehydrogenase in animals exposed to

250 ppm. Bronchoalveolar lavage fluid 3 from animals receiving

175 ppm contained greater amounts of protein, albumin, LDH, and 4 angiotensin converting enzyme activities than unexposed controls and all treated groups had increased 5 numbers of neutrophilic leucocytes. Dose related increases in severity of centriacinar catarrhal 6 pneumonitis, macrophage influx, and neutrophilic leucocytes were observed on histopathological 7 examination of the lungs. Edema occurred at

250 ppm, subpleural hemorrhaging at

400 ppm, and 8 desquamation of the bronchiolar epithelium was seen at

600 ppm. 9 10 3.1.3. Guinea Pigs 11 12 Hine et al. ( 1970) also studied the effects of varying concentration and duration of exposure 13 in the guinea pig ( strain not specified). Animals ( n = 2 6) were exposed to 5 200 ppm NO 2 for 30 14 minutes up to 8 hours. At concentrations of

40 ppm, signs of toxicity included lacrimation, 15 reddening of the conjunctivae, and increased respiration which became labored and difficult as the 16 intoxication increased. Mortalities were first observed at 50 ppm for 1 hour ( Table 3). Terminally, 17 respiration became gasping and spasmodic and lung edema was observed at necropsy. Histological 18 observations of the lungs following deaths were characterized by bronchiolitis, desquamated bronchial 19 epithelium, infiltration by polymorphonuclear cells, and edema. 20 21 To determine the sensitivity of adult and neonate animals to NO 2 inhalation, Duncan Hartley 22 guinea pigs aged 5 60 days were exposed continuously for 3 days to 2 or 10 ppm ( Azoulay Dupuis 23 et al., 1983). A total of 17 27 animals were studied in each age group and litter exposures prior to 24 weaning included the dam. At 10 ppm, clinical signs of toxicity in adults over 45 days old included 25 moving with difficulty, reduced food and water consumption, and hyperventilation. Body weight 26 gains were decreased until 21 days and body weights were reduced after 45 days in all exposed 27 animals. These effects were most pronounced in the dams. Mortality in the high exposure group 28 increased with age with 4% of 5 day olds, up to 60% of 55 day olds, and 67% of dams dying. 29 Further, most of the older animals died after the first 24 hours of exposure whereas the younger 30 animals died later in the 3 day period. At 2 ppm, lung histopathology was normal until animals were 31 45 days of age when thickening of the alveolar walls, infiltration by PMNs, and alveolar edema were 32 observed; in dams bronchioles were devoid of cilia in some areas. At 10 ppm, guinea pigs of all ages 33 were affected by these changes which were more pronounced in older animals. 34 35 3.1.4. Rats 36 37 Five, 15, 30, and 60 minute LC 50 values for the male rat ( 100 120 g; strain not specified; 38 n = 10) are 416, 201, 162, and 115 ppm, respectively. Clinical signs of toxicity included severe 39 respiratory distress, eye irritation, 10 15% body weight suppression, and death; time to death varied 40 from 30 minutes to 3 days. Gross pathology revealed darkened areas about the surface of the lungs, 41 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 22 and, in some instances, purulent nodules involving the entire lungs of some of the survivors ( Carson 1 et al., 1962). 2 3 An older study ( Gray et al., 1954) reported LC 50 values for male rats ( 200 300 g; strain not 4 specified; n = 10) of 1445 ppm for 2 minutes, 833 ppm for 5 minutes, 420 ppm for 15 minutes, 174 5 ppm for 30 minutes, 168 ppm for 60 minutes, and 88 ppm for 240 minutes. Deaths were attributed 6 to pulmonary edema. The differences in LC 50 values between this study and Carson et al. ( 1962) may 7 be due to the differences in size and age of the rats used in each. 8 9 Meulenbelt et al. ( 1992a, b) investigated the effects of both concentration and duration of 10 exposure in Wistar rats. The effect of concentration was studied by exposing 6 9 rats/ group to 25, 11 75, 125, 175 or 200 ppm NO 2 for 10 minutes. No signs of toxicity were observed at 25 ppm. 12 Stertorous respirations were heard in animals exposed to 175 and 200 ppm. Rats exposed to

75 13 ppm had significantly increased lung weight, and subpleural hemorrhages and pale discolorations of 14 the lung were observed grossly. Histologically, the lungs from these animals showed atypical 15 pneumonia, edema, focal desquamation of the terminal bronchiolar epithelium, increased numbers of 16 macrophages and neutrophilic leucocytes, and interstitial thickening of the centriacinar septa ( 175 and 17 200 ppm only) with the severity increasing at the higher concentrations. One rat died 14 20 hours 18 after exposure in both the 175 ppm and 200 ppm groups. Biochemical changes in bronchoalveolar 19 lavage fluid included concentration dependent increases in protein and albumin concentrations, 20 angiotensin converting enzyme activity,   glucuronidase activity, and neutrophilic leukocytes. 21 22 Duration of exposure was investigated by exposing 6 rats/ group to either 175 ppm for 10, 20, 23 or 30 minutes or to 400 ppm for 5, 10, or 20 minutes ( Meulenbelt et al., 1992a, b). Stertorous 24 respirations were heard in animals for all exposure times of both concentrations and lung weights 25 were significantly higher than the controls at all exposure durations. At 175 ppm, 5/ 6 rats died in the 26 20 and 30 minute groups and at 400 ppm, 6/ 6 rats died in the 10 and 20 minute groups. Necropsy 27 revealed foamy, seroanguinous fluid in the trachea, subpleural bleeding, and pale discoloration. 28 Histological alterations were similar to those described above. Methemoglobin levels, measured after 29 exposure to 175 ppm for 10 minutes, were not elevated, but plasma nitrate levels were significantly 30 higher than controls. 31 32 Hine et al. ( 1970) also studied the effects of varying concentration and duration of exposure 33 in Long Evans rats. Animals ( n= 4 31) were exposed to 5 250 ppm NO 2 for durations of 30 minutes 34 up to 24 hours. At concentrations of

40 ppm, signs of toxicity included lacrimation, reddening of 35 the conjunctivae, and increased respiration which became labored and difficult as the intoxication 36 increased. Mortalities were first observed at 50 ppm for 24 hours ( Table 3). Terminally, respiration 37 became gasping and spasmodic and lung edema was observed at necropsy. Histological observations 38 of the lungs following deaths were characterized by bronchiolitis, desquamated bronchial epithelium, 39 infiltration by polymorphonuclear cells, and edema. 40 41 3.1.5. Mice 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 23 BALB/ c mice ( n = 5 7) were exposed to 5, 20, or 40 ppm NO 2 for 12 hours ( Hidekazu and 1 Fujio, 1981). Body weights were markedly decreased 1 and 2 days after exposure to 20 and 40 ppm 2 and 3/ 38 ( 7.8%) animals exposed to 40 ppm died within 2 days. 3 4 Hine et al. ( 1970) studied the effects of varying concentration and duration of exposure in 5 Swiss Webster mice. Animals ( n= 5 14) were exposed to 5 250 ppm NO 2 for durations of 30 6 minutes up to 24 hours. At concentrations of

40 ppm, signs of toxicity included lacrimation, 7 reddening of the conjunctivae, and increased respiration which became labored and difficult as the 8 intoxication increased. Mortalities were first observed at 50 ppm for 24 hours ( Table 3). Terminally, 9 respiration became gasping and spasmodic and lung edema was observed at necropsy. Histological 10 observations of the lungs following deaths were characterized by bronchiolitis, desquamated bronchial 11 epithelium, infiltration by polymorphonuclear cells, and edema. 12 13 14 Table 3: Summary of NO2 Mortality for Five Species 15 Conc. 16 ( ppm) 17 Time ( hr) C × t ( ppm hr) Rat Mouse Guinea Pig Rabbit Dog 50 18 1 6 24 50 300 1200 0/ 17 0/ 12 3/ 10 0/ 5 0/ 5 5/ 10 1/ 6 4/ 6  0/ 4 0/ 4  0/ 1 0/ 2

75 19 1 2 4 8 75 150 300 600 3/ 31 1/ 12 7/ 12 12/ 12 1/ 6 2/ 6 5/ 6 6/ 6 1/ 4 3/ 4 2/ 4 4/ 4 1/ 8 0/ 6 2/ 8 6/ 8 0/ 2 0/ 2 1/ 3 1/ 4 100 20 0.5 2 4 8 50 200 400 800 0/ 5 8/ 8 29/ 29  2/ 10 13/ 14 10/ 10 10/ 10 1/ 2 3/ 4   1/ 3 2/ 4 3/ 4  0/ 2 1/ 3 2/ 2

150 21 0.5 1 2 4 75 150 300 600 2/ 10 10/ 13 10/ 12 4/ 4     3/ 4  3/ 3   1/ 6  3/ 4  2/ 3

200 22 0.08 0.17 0.33 0.50 16.7 33.3 66.7 100 6/ 12 8/ 12 5/ 5 4/ 4 4/ 6 6/ 6 6/ 6  2/ 2    0/ 2 1/ 2 2/ 4    2/ 2

23 Data from Hine et al., 1970, p. 206; deaths generally occurred within 2 8 hours after exposure and the majority within 24 hours. 24 25 26 3.2. Nonlethal Toxicity 27 3.2.1. Monkeys 28 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 24 Squirrel monkeys ( n = 2 6/ group) were exposed to 10 50 ppm NO 2 for 2 hours with 1 respiratory function monitored during exposure ( Henry et al., 1969). Exposure to 35 or 50 ppm 2 resulted in a markedly increased respiratory rate and decreased tidal volume during exposures which 3 returned to normal by the seventh day post exposure. Only slight effects on respiratory function were 4 noted at 15 and 10 ppm. Mild histopathological changes in the lungs were noted after exposure to 5 10 and 15 ppm, however, marked changes in lung structure were observed after exposure to 35 and 6 50 ppm. At 35 ppm, areas of the lung were collapsed with basophilic alveolar septa, in other areas 7 the alveoli were expanded with septal wall thinning, and the bronchi were moderately inflamed with 8 some proliferation of the surface epithelium. At 50 ppm, extreme vesicular dilatation of alveoli or 9 total collapse was observed, lymphocyte infiltration was seen with extensive edema, and surface 10 erosion of the epithelium of the bronchi was observed. In addition to the effects on the lungs, 11 interstitial fibrosis ( 35 ppm) and edema ( 50 ppm) of cardiac tissue, glomerular tuft swelling in the 12 kidney ( 35 and 50 ppm), lymphocyte infiltration in the kidney and liver ( 50 ppm), and congestion and 13 centrilobular necrosis in the liver ( 50 ppm) were observed. 14 15 3.2.2. Dogs 16 17 Carson et al. ( 1962) conducted a series of experiments on dogs ( strain not specified; n = 2) 18 at target concentrations of NO 2 approximately 50% and 25% of the LC 50 for the rat ( see Sec. 3.1.5). 19 The actual analyzed concentrations varied slightly, but were within 10% of target. For the 50% 20 concentrations, dogs exposed to 164 ppm for 5 minutes, 85 ppm for 15 minutes, or 53 ppm for 60 21 minutes had some respiratory distress during exposure, a mild cough, and eye irritation all of which 22 cleared within two days after exposure. At approximately 25% of the rat LC 50, dogs exposed to 125 23 ppm for 5 minutes, 52 ppm for 15 minutes, or 39 ppm for 60 minutes showed only mild sensory 24 effects. No gross or microscopic lesions were noted in any dog. 25 26 Greenbaum et al. ( 1967) exposed mongrel dogs ( n = 1) to 0.1% ( 1000 ppm) NO 2 for 136 27 minutes or to 0.5% ( 5000 ppm) for 5 45 minutes. A concentration of 0.1% did not cause death and 28 the one dog exposed to this concentration remained in good condition throughout the exposure. 29 Exposures to 0.5% for 15 and 22 minutes were not lethal but resulted in respiratory distress which 30 gave rise to anxiety for about 2 hours then resolved without therapy. Histopathologic examination 31 of the lungs was not performed after sacrifice. 32 33 No treatment related changes in behavior or clinical signs were observed in mongrel dogs ( n 34 = 1) exposed to 10 40 ppm NO 2 for 6 hours ( Henschler and Lütke, 1963). 35 36 Mongrel dogs ( number of animals not stated) exposed to 20 ppm NO 2 for up to 24 hours 37 showed minimal signs of irritation and changes in behavior. Microscopic lesions were described as 38 questionable evidence of lung congestion and interstitial inflammation for up to 48 hours 39 postexposure ( Hine et al., 1970). 40 41 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 25 Pulmonary ultrastructural changes were examined in beagle dogs ( n = 1) exposed to 3 16 ppm 1 NO 2 for 1 hour ( Dowell et al., 1971). Intraalveolar edema occurred in most dogs exposed to

7 ppm 2 and was associated with impaired surfactant activity and lung compliance. Ultrastructural alterations 3 included wide spread bleb formation, loss of pinocytic vesicles, and mitochondrial swelling of 4 endothelial cells. Exposure to 3 ppm resulted in bleb formation in the alveolar endothelium ( observed 5 by EM) without biochemical or physiological changes. 6 7 3.2.3. Rabbits 8 9 Rabbits ( strain not specified; number of animals not given) exposed to 20 ppm NO 2 for up to 10 24 hours showed minimal signs of irritation and changes in behavior. Microscopic lesions were 11 described as questionable evidence of lung congestion and interstitial inflammation for up to 48 hours 12 postexposure ( Hine et al., 1970). 13 14 Rabbits exposed to 10 ppm NO 2 for 2 hours showed accelerated alveolar particle clearance 15 ( Vollmuth et al., 1986) and altered pulmonary arachidonic acid metabolism ( Schlesinger et al., 1990). 16 Continuous exposure of rabbits to 3.6 ppm NO 2 for 6 days did not cause morphological changes in 17 the lungs ( Hugod, 1979). 18 19 3.2.4. Sheep 20 21 Lung mechanics, hemodynamics, and blood chemistries were assessed in crossbred sheep ( n 22 = 5 6) exposed by nose only or lung only ( to mimic mouth breathing) to 500 ppm NO 2 for 15 23 minutes; in another group also exposed by lung only, bronchoalveolar lavage fluid content was 24 examined after a 20 minute exposure to 500 ppm ( Januszkiewicz and Mayorga, 1994). No changes 25 in hemodynamics or blood chemistries occurred in either group. Mean inspired minute ventilation 26 was significantly increased, resulting in increased breathing rate and decreased mean tidal volume, 27 in the lung only exposure group, but not the nose only group. Both nose only and lung only 28 exposure groups had significantly increased lung resistance and decreased dynamic lung compliance. 29 Histopathologic examination of tissue from the lung only exposed group revealed exudative fluid 30 distributed in a patchy lobular pattern with mild neutrophil infiltration; little evidence of exudation 31 was seen in the nose only exposed group. Epithelial cell number and total protein in bronchoalveolar 32 lavage fluid were significantly increased in the NO 2 exposed animals while macrophage number was 33 decreased. 34 35 Airway reactivity to aerosolized carbachol was evaluated in crossbred sheep ( n = 4 10) 36 exposed to 7.5 or 15 ppm NO 2 for 2 hours ( Abraham et al., 1980). Group means for pulmonary 37 resistance, bronchial reactivity to carbachol, or static lung compliance were not different from 38 controls at either concentration. However, following exposure to 7.5 ppm NO 2, 5 of 9 animals 39 showed an increase in pulmonary resistance after carbachol exposure of 57% above baseline and 40 following exposure to 15 ppm NO 2, 9 of 10 animals responded with either bronchoconstriction or 41 hyperreactivity. In a concurrent experiment, sheep were exposed to 15 ppm NO 2 for 4 hours 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 26 ( Abraham et al., 1980). Mean pulmonary resistance was significantly increased from the preexposure 1 value, but there were no changes in pulmonary hemodynamics or clinical signs of distress. 2 3 3.2.5. Guinea Pigs 4 5 Guinea pigs ( strain not specified; number of animals not given) exposed to 20 ppm NO 2 for 6 up to 24 hours showed minimal signs of irritation and changes in behavior. Microscopic lesions were 7 described as questionable evidence of lung congestion and interstitial inflammation for up to 48 hours 8 postexposure ( Hine et al., 1970). Guinea pigs exposed to 9 and 13 ppm for 2 hours or to 5.2 and 6.5 9 ppm for 4 hours had significantly increased respiratory rate and decreased tidal volume with complete 10 recovery after cessation of exposure ( Murphy et al., 1964). 11 12 Hartley guinea pigs ( n = 5 16) maintained on an ascorbic acid deficient diet had increased lung 13 lavage fluid protein following exposure to 4.8 ppm NO 2 for 3 hours and increased wet lung weight, 14 increased nonprotein sulfhydryl and ascorbic acid content of the lungs, and decreased   tocopherol 15 content of the lungs following exposure to 4.5 ppm for 16 hours. These changes were not seen in 16 animals maintained on normal guinea pig diets ( Hatch et al., 1986). 17 18 Guinea pigs ( strain not specified; n = 12 18) were exposed to 20, 40, or 70 ppm NO 2 for 30 19 minutes followed by a 30 minute exposure to aerosolized albumin; this regimen was repeated 5 7 20 times at intervals of several days ( Matsumura, 1970). During the first exposure to 70 ppm, labored 21 breathing, though not severe, was observed in " some"' animals, but was not seen with subsequent 22 exposures. Immediately after the fifth exposure to antigen, one half of the animals in the 70 ppm 23 group showed enhanced airway sensitization ( anaphylactic attacks). No effects were seen at 20 or 24 40 ppm. 25 26 Changes in airway responsiveness to histamine were investigated in Hartley guinea pigs 27 ( number of animals not given) exposed to 7 146 ppm NO 2 for 1 hour ( Silbaugh et al., 1981). 28 Pulmonary function measurements and histamine challenge tests were performed 2 hours before and 29 at about 10 minutes, 2 and 19 hours after NO 2 exposure. At 10 minutes after exposure, increased 30 sensitivity to histamine occurred at concentrations

40 ppm but returned to baseline thereafter. 31 Concentration related significantly increased breathing frequencies and decreased tidal volumes were 32 measured at 10 minutes ( exact concentrations not specified) and remained correlated with 33 concentration at 2 and 19 hours. 34 35 3.2.6. Hamsters 36 37 Syrian golden hamsters ( n = 5) were administered 28 ppm NO 2 for 6, 24, or 48 hours and the 38 histopathological changes in the lungs were examined by light and electron microscopy ( Case et al., 39 1982; Gordon et al., 1983). The bronchiolar epithelium showed ciliary loss and surface membrane 40 damage, loss of ciliated cells, and epithelial flattening at 24 and 48 hours and epithelial hyperplasia, 41 nonciliated cell hypertrophy, and loss of tight junctions between type I pneumocytes at 48 hours. 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 27 3.2.7. Ferrets 1 2 Weanling domestic ferrets ( n = 4 6), 6 weeks of age, were exposed to 5, 10, 15, or 20 ppm 3 NO 2 for 4 hours ( Rasmussen, 1992). A transient inflammatory response was evident as a significantly 4 increased number of neutrophils in the lavage fluid up to 48 hours postexposure to all concentrations. 5 Morphometrically, a dose related decreased alveolar size and thickened alveolar walls indicative of 6 exposure were observed in the lungs. 7 8 3.2.8. Rats 9 10 Pulmonary injury from NO 2 as indicated by increases in lung weights was assessed in male 11 Fischer 344 rats ( n = 6 12) following exposure to 10, 25, or 50 ppm for 5, 15, or 30 minutes or to 12 100 ppm for 5 or 15 minutes ( Stavert and Lehnert, 1990). No significant changes in lung weight 13 occurred in rats exposed to 10 ppm for 30 minutes or to 25 50 ppm for up to 15 minutes. Significant 14 increases in lung wet weight and right cranial lobe dry weight were found following exposure to 50 15 ppm for 30 minutes or to 100 ppm for 5 and 15 minutes. However, histological evidence of lung 16 injury was seen in animals exposed to 25 ppm for 30 minutes, 50 ppm for

5 minutes, and 100 ppm 17 for 5 and 15 minutes. This was described as accumulation of fibrin, increased numbers of 18 polymorphonuclear leukocytes and macrophages, extravasated erythrocytes, and type II pneumocyte 19 hyperplasia the severity of which increased with concentration and duration of exposure. 20 21 In a more expanded study, Lehnert et al. ( 1994) determined that exposure concentration was 22 more important than exposure duration in the severity of lung injury. Male Fischer 344 rats ( n = 8 23 12) were exposed to 25, 50, 75, 100, 150, 200, or 250 ppm NO 2 for durations ranging from 5 30 24 minutes. Lung wet weights were significantly increased following exposures to

150 ppm for 5 25 minutes, 100 ppm for 15 minutes, and 75 ppm for 30 minutes and further increases were observed 26 as exposure duration increased. The pulmonary edematous response to a given concentration was 27 not proportional to duration, however, increasing concentrations produced proportional increases in 28 lung wet weight when similar exposure durations were compared. Histologically, fibrin and type II 29 cell hyperplasia were observed following 5 minute exposures to

50 ppm the severity of which 30 increased proportionally to concentration. As further confirmation of concentration dependent lung 31 injury, rats were exposed to 1 minute bursts of 500 2000 ppm. The severity of the resulting 32 pulmonary edema ( as measured by lung wet weight) was directly proportional to exposure 33 concentration. The authors concluded that brief exposures to the high concentrations of NO 2 are 34 more injurious than longer duration exposures to lower concentrations. Dietary taurine ( an 35 antioxidant) was not protective against the increase in lung wet weight and exercise potentiated the 36 severity of the pulmonary edema. 37 38 The concentration dependent response of the lung to NO 2 was confirmed in another study in 39 which Sprague Dawley rats ( n = 5 6) were exposed to 3.6 14.4 ppm for durations of 6 24 hours/ day 40 for 3 days ( Gelzleichter et al., 1992). Increases in protein content and cell types in lavage fluid 41 demonstrated that the magnitude of lung injury was a function of exposure concentration. 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 28 Carson et al. ( 1962) conducted a series of experiments at NO 2 concentrations approximating 1 50%, 25%, and 15% of the rat LC 50 levels. At the 50% level, rats ( strain not specified; n = 30) 2 exposed to 190 ppm for 5 minutes, 90 ppm for 15 minutes, or 72 ppm for 60 minutes showed signs 3 of severe respiratory distress and eye irritation lasting about two days; lung to body weight ratios 4 were significantly increased during the first 48 hours after exposure. Pathological examination 5 showed darkened areas of the lungs, pulmonary edema, and an increased incidence of chronic murine 6 pneumonia. Rats exposed to 104 ppm for 5 minutes, 65 ppm for 15 minutes, and 28 ppm for 60 7 minutes ( about 25% of the LC 50 s) showed some respiratory distress or mild signs of nasal irritation 8 during exposure but lung to body weight ratios were increased only at the 104 and 65 ppm levels. 9 No gross lesions were observed, but pulmonary edema was seen microscopically. No adverse clinical 10 signs of toxicity or pathological changes were seen in rats exposed at 15% of the LC 50 ( 74 and 33 11 ppm for 5 and 15 minutes, respectively). 12 13 Histological changes in the lungs of male rats ( strain not specified; number of animals not 14 given) exposed to 17 ppm NO 2 were examined during continuous exposure ( Stephens et al., 1972). 15 After 2 hours of exposure, there was some precapillary and postcapillary engorgement in the alveoli. 16 Loss of cilia and occasional alveolar type I cell swelling were detectable by 4 hours, the terminal 17 bronchiolar epithelium had become uniform by 16 hours, maximal macrophage numbers were reached 18 by 24 hours, cellular hypertrophy had begun by 48 hours, and mitotic figures became more prevalent 19 in the epithelium of the terminal bronchiole between 16 and 48 hours. Type I alveolar cells appeared 20 to be the most sensitive to NO 2 insult. 21 22 Results similar to those described above were obtained in a morphological study of the Wistar 23 rat lung ( number of animals not given) following exposure to 20 ppm NO 2 for 20 hours ( Hayashi et 24 al., 1987). Cytoplasmic blebbing occurred in a small number of type I cells immediately after 25 exposure. Swelling and hyperplasia of type II cells and pinocytotic vesicles of endothelial cells in 26 capillaries followed by interstitial edema in the alveolar walls were observed between days 5 15 27 postexposure. Twenty days after exposure the lesions lessened and the lungs appeared normal after 28 35 days. Other studies have confirmed alveolar and interstitial edema, bronchiolitis, bronchiolar 29 epithelial cell hyperplasia, loss of cilia, necrosis of type I cells, and/ or type II cell hyperplasia 1 3 days 30 after exposure to 26 ppm NO 2 for 24 hours ( Schnizlein et al., 1980; Hillam et al., 1983) or to 20 ppm 31 for 24 hours ( Rombout et al., 1986). 32 33 Long Evans rats ( number of animals not given) exposed to 20 ppm NO 2 for up to 24 hours 34 showed minimal signs of irritation and changes in behavior. Microscopic lesions were described as 35 questionable evidence of lung congestion and interstitial inflammation for up to 48 hours 36 postexposure ( Hine et al., 1970). 37 38 The effects of NO 2 on the lung were compared between Sprague Dawley neonatal and adult 39 rats ( number of animals not given). Animals, 1 40 days old, were continuously exposed to 14 ppm 40 for 24, 48, or 72 hours. Prior to weaning ( 20 days old), exposure resulted in only minor injury and 41 loss of cilia from epithelial cells lining the terminal airways. Subsequent to weaning, there was a 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 29 progressive increase in lung injury with maximum response reached at about 35 days of age ( Stephens 1 et al., 1978). 2 3 In a similar study to determine the sensitivity of adult and neonate animals to NO 2 inhalation, 4 Wistar rats ( number of animals not given) aged 5 60 days were exposed continuously for 3 days to 5 2 or 10 ppm ( Azoulay Dupuis et al., 1983). Litter exposures prior to weaning included the dam. No 6 clinical signs of toxicity or deaths were observed in animals of any age except for body weight loss 7 in dams of the 10 ppm group. At 2 ppm, lung histopathology was normal in all animals. At 10 ppm 8 in animals 45 days old and older, fibrinous deposits were observed in the alveoli and the tracheal and 9 bronchiolar epithelia were occasionally devoid of cilia. 10 11 Alterations in lavage fluid have been assessed in male Long Evans rats ( n = 6) following 12 exposure to 10, 20, 30, or 40 ppm for 4 hours. Cell free lavage fluid contained elevated lactate 13 dehydrogenase ( LDH), malate dehydrogenase ( MDH), isocitrate dehydrogenase ( IDH), glucose 6 14 phosphate dehydrogenase ( GDH), acid phosphatase ( AP), and aryl sulfatase ( AS) after exposure to 15

30 ppm. Total protein and sialic acid were increased after exposure to

20 ppm. Protein, AP, and 16 sialic acid concentrations were similar to plasma indicating transudation into the airways ( Guth and 17 Mavis, 1985). The increases in LDH, MDH, and GDH were significantly attenuated in animals 18 maintained on diets providing 1000 mg/ kg of   tocopherol, suggesting that lipid peroxidation is 19 involved in NO 2 induced lung injury ( Guth and Mavis, 1986). Antioxidants in the lung were depleted, 20 lipid peroxidation products were elevated, and total cell count in BALF and alveolar macrophage 21 count were decreased while epithelial cell count was increased following exposure of male Sprague 22 Dawley rats ( n = 5) to 200 ppm for 15 minutes ( Elsayed et al., 2002). Another study found changes 23 in fatty acid composition of alveolar lavage phospholipids following exposure of Wistar rats ( n = 6) 24 to 20 ppm NO 2 for 12 hours ( Kobayashi et al., 1984). Increases in lavageable protein, 25 polymorphonuclear lymphocytes, and alveolar macrophages were also observed following exposure 26 of male Fischer 344 rats ( n not given) to 100 ppm for 15 minutes ( Lehnert et al., 1994). 27 28 Changes in minute ventilation, V E, were measured in male Fischer 344 rats ( n = 12) following 29 exposure to 100, 300, or 1000 ppm NO 2 for 1 20 minutes ( Lehnert et al., 1994). In general, 30 reductions in V E were greater with the higher concentrations. For example, reductions of about 7% 31 and 15% were measured during 15 and 20 minute exposure to 100 ppm, while reduction of about 32 20% and 28% were measured during 1 and 2 minute exposures to 1000 ppm. Similarly, male 33 Sprague Dawley rats ( n = 5) exposed to 200 ppm for 15 minutes showed a decrease in minute 34 ventilation that was due to a decline in tidal volume but not in frequency of breathing ( Elsayed et al., 35 2002). 36 37 Male Porton rats ( n = 4) were exposed to an atmosphere of oxides of nitrogen that was 38 produced by mixing nitrogen dioxide and nitric oxide ( Brown et al., 1983). Exposures were to 518 39 ppm for 5 minutes or to 1435 ppm for 1 minute. No clinical signs of toxicity were observed during 40 exposure to either concentration but " stertorous respirations" appeared within 24 hours. 41 Histologically, initial lung damage showed thickening and blebbing of the alveolar epithelium followed 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 30 by a latent period of about 6 hours after which development of edema of the interstitium and alveolar 1 septum was observed. The early changes were attributed to a direct oxidant effect. 2 3 Changes in lung immunity have been described as increased specific IgE, IgA, and IgG titers 4 following exposure to 87 ppm for 1 hour ( Siegel et al., 1997) or 5 ppm for 3 hours ( Gilmour, 1995), 5 increased number of IgG anti sheep red blood cell antibody forming cell in the lung associated lymph 6 nodes ( Schnizlein et al., 1980), and cell proliferation in the spleen and thoracic lymph nodes ( Hillam 7 et al., 1983) following exposure to 26 ppm for 24 hours. 8 9 3.2.9. Mice 10 11 Swiss Webster mice ( number of animals not given) exposed to 20 ppm NO 2 for up to 24 12 hours showed minimal signs of irritation and changes in behavior. Histologically, there was 13 questionable evidence of lung congestion and interstitial inflammation for up to 48 hours 14 postexposure ( Hine et al., 1970). The voluntary running activity of mice on an activity wheel was 15 17% and 80% of preexposure levels during 6 hour exposures to 7.7 and 20.9 ppm, respectively 16 ( Murphy et al., 1964). 17 18 The alveolar septum from two female NMRI mice was examined microscopically thirty six 19 hours after exposure to 35 ppm for 6 hours ( Dillmann et al., 1967). Morphometric measurements 20 found that the arithmetic mean thickness of the alveoli was approximately 1.5x that of unexposed 21 controls. No changes in the number and types of cells present were observed and no interstitial 22 edema was seen with ultrastructure examination by electron microscopy. 23 24 Male CD 1 mice ( n = 5 9) were exposed to 50 140 ppm NO 2 for 1 hour and biochemical and 25 histological responses assessed immediately and 48 hours after exposure ( Siegel et al., 1989). 26 Immediately after exposure to 140 ppm, cell death was visible in the terminal bronchioles and there 27 were significant increases in protease inhibitor activity, pulmonary protein, and lung wet weight. Two 28 days after exposure to 140 ppm, the histological damage was exacerbated with complete obliteration 29 of the alveolar structure, progressive edema and congestion of the lungs, hypertrophy and hyperplasia 30 of the epithelial cells, and increased numbers of intraalveolar macrophages and neutrophils. Also two 31 days after exposure, there were dose related increases in   glucuronidase, lactate dehydrogenase, and 32 choline kinase activities as well as increased protease inhibitor activity, pulmonary protein, and lung 33 wet weight. 34 35 To examine the effects of NO 2 on gaseous exchange in the lung, JCL: ICR mice ( n = 6) were 36 exposed to 5, 10, or 20 ppm for 24 hours ( Suzuki et al., 1982). Significantly increased lung wet 37 weight and lung water content occurred at 10 and 20 ppm. In animals exposed to 5 ppm, the gaseous 38 exchange and metabolic rate of O 2 and CO 2 were accelerated while in animals exposed to 10 and 20 39 ppm, gaseous exchange in the lung was inhibited. 40 41 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 31 Continuous exposure of C56Bl/ 6 mice ( n = 60) to 20 ppm NO 2 for four days resulted in 1 significantly decreased food consumption and body weight, but no deaths ( Bouley et al., 1986). 2 3 3.3. Developmental/ Reproductive Toxicity 4 5 The postnatal effects of prenatal exposure to NO 2 were investigated ( Tabacova et al., 1985). 6 Pregnant Wistar rats ( n = 20) were exposed to 0.265, 0.053, 0.53, or 5.3 ppm NO 2 for 6 hours/ day 7 throughout pregnancy. Maternal effects were not reported or discussed. Pup viability and body 8 weight of the 5.3 ppm group were significantly ( p  0.05) less than the controls on lactation day 21. 9 Exposure to

0.53 ppm resulted in developmental delays and exposure to

0.053 ppm caused 10 disturbances in neuromotor development. Also at the two highest concentrations, hexobarbital 11 sleeping time was increased in the offspring and correlated with altered biochemical parameters in the 12 liver. 13 14 3.4. Genotoxicity 15 16 Three week old male Sprague Dawley rats were exposed by inhalation to 8, 15, 21, or 27 17 ppm NO 2 for 3 hours, maintained overnight before sacrifice, and lung cells isolated. At 18 concentrations of

15 ppm, there was a concentration related increase in mutation to ouabain 19 resistance in lung cells. Concentration dependent increases in chromosome aberrations were 20 observed from exposures to 8 and 27 ppm NO 2, the only concentrations analyzed for aberrations 21 ( Isomura et al., 1984). 22 23 3.5. Subchronic and Chronic Toxicity/ Carcinogenicity 24 25 Respiratory effects in humans from long term environmental exposures to NO 2 have been 26 discussed with the epidemiology studies in Section 2.2.2. 27 28 The effect of NO 2 on promotion of lung tumorigenesis induced by N bis( 2 hydroxypropyl) 29 nitrosamine ( BHPN) was investigated in male Wistar rats ( Ichinose et al., 1991). Animals were given 30 a single intraperitoneal injection of 0.5 g BHPN/ kg body weight at 6 weeks of age and exposed to 31 0.04, 0.4, or 4.0 ppm NO 2 for 17 months. The incidence of pulmonary tumors in rats exposed to 32 BHPN plus 4 ppm NO 2 was 12.5% ( n. s.) with adenomas found in 4/ 40 rats ( 10%) and 33 adenocarcinomas found in 1/ 40 rats ( 2.5%). One adenoma was found in the control group ( 2.5%) 34 and one in the 0.04 ppm group, but none in the 0.4 ppm group. In addition, marked bronchiolar 35 mucosal hyperplasia was found in 17/ 40 rats ( 42.5%, p  0.001) in the BHPN plus 4.0 ppm NO 2 36 group. 37 38 3.6. Summary 39 40 Five to 60 minute LC 50 values for the rat ranged from 416 to 115 ppm, respectively in one 41 study ( Carson et al., 1962) and from 833 to 168 ppm in another study ( Gray et al., 1954). The 15 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 32 minute LC 50 for rabbits was 315 ppm ( Carson et al., 1962). In a study using varying concentration 1 and duration of exposure, the first mortalities were observed in dogs at 75 ppm for 4 hours, in rabbits 2 at 75 ppm for 1 hour, in guinea pigs at 50 ppm for 1 hour, and in rats and mice at 50 ppm for 24 3 hours ( Hine et al., 1970). Histological alterations of the lungs following death included bronchiolitis, 4 desquamated bronchial epithelium, infiltration by polymorphonuclear cells, and edema. Enhanced 5 susceptibility to infection was shown in monkeys following exposure to 50 ppm for 2 hours ( Henry 6 et al., 1969) and in mice following exposure to 2 or 3.5 ppm for 3 hours ( Ehrlich, 1978). 7 8 Pulmonary edema and histological alterations induced by exposure to NO 2 have been 9 characterized in dogs, sheep, guinea pigs, hamsters, rats, and mice. Numerous studies in rats have 10 confirmed alveolar and interstitial edema, bronchiolitis, bronchiolar epithelial cell hyperplasia, loss 11 of cilia, necrosis of type I cells, and/ or type II cell hyperplasia 1 3 days after exposure to 26 ppm NO 2 12 for 24 hours ( Schnizlein et al., 1980; Hillam et al., 1983) or to 20 ppm for 20 ( Hayashi et al., 1987) 13 or 24 hours ( Rombout et al., 1986). 14 15 Neonates appear less sensitive to NO 2 than adult animals with progressive increases in lung 16 injury and deaths seen in older rats and guinea pigs ( Stephens et al., 1978; Azoulay Dupuis et al., 17 1983). 18 19 20 4. SPECIAL CONSIDERATIONS 21 4.1. Metabolism and Disposition 22 23 Total respiratory tract absorption of NO 2 by humans exposed to 0.29 7.2 ppm for  30 24 minutes during quiet respiration and during exercise has been measured at 81 90% and 91 92%, 25 respectively in healthy adults and 72% and 87%, respectively in asthmatics ( U. S. EPA, 1993). In 26 monkeys exposed to 0.30 0.91 ppm NO 2 for < 10 minutes, 50 60% of the inspired gas has been 27 shown to be retained during quiet respiration with distribution throughout the lungs ( Goldstein et al., 28 1977). While the isolated rat lung, ventilated with 5 ppm for 90 minutes, retained 36% of the NO 2 29 ( Postlethwait and Mustafa, 1981), the majority of labeled NO 2 ( exposure parameters not specified) 30 was retained by the upper respiratory tract of the rat ( Russell et al., 1991). 31 32 Pulmonary absorption of NO 2 has been studied using in vivo and in vitro models. Uptake 33 appears to be governed by the reaction between inhaled NO 2 and constituents of the pulmonary 34 surface lining layer to form nitrite ( Postlethwait and Bidani, 1990, 1994; Saul and Archer, 1983). 35 NO 2 uptake is saturable with absorption proportional to inspired dose ( Postlethwait and Bidani, 1994) 36 and has been shown to increase as temperature increases to a maximum of 10.6 µ g NO 2/ min in an 37 isolated lung model ( Postlethwait and Bidani, 1990). The predominant reaction in the lungs is still 38 debated and may involve hydrogen abstraction by readily oxidizable tissue components such as 39 proteins and lipids to form nitrous acid and the nitrite radical ( Postlethwait and Bidani, 1994) or it 40 may be reaction with water to form nitrous and nitric acids ( Goldstein et al., 1977). 41 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 33 Distribution of inhaled NO 2 or its metabolites is via the blood stream ( Goldstein et al., 1977). 1 Nitrite formed in the lungs is oxidized to nitrate by interactions with RBCs after diffusion into the 2 vascular space ( Postlethwait and Mustafa, 1981). Exposure of mice to 40 ppm NO 2 produced slight 3 ( 0.2%) nitrosylhemoglobin but no methemoglobin ( Oda et al., 1980) and an increase in both nitrite 4 and nitrate that reached equilibrium in 10 and 30 minutes, respectively ( Oda et al., 1981). After 5 cessation of exposure, nitrite had a half life of several minutes and nitrate had a half life of about 1 6 hour ( Oda et al., 1981). Urinary excretion of nitrate has been shown to be linearly related to the NO 2 7 concentration administered via inhalation ( Saul and Archer, 1983). 8 9 4.2. Mechanism of Toxicity 10 11 NO 2 is an irritant to the mucous membranes and may cause coughing and dyspnea during 12 exposure. After less severe exposure, symptoms may persist for several hours before subsiding 13 ( NIOSH, 1976). With more severe exposure, pulmonary edema ensues with signs of chest pain, 14 cough, dyspnea, cyanosis, and moist rales heard on auscultation ( NIOSH, 1976; Douglas et al., 15 1989). Death from NO 2 inhalation is caused by bronchospasm and pulmonary edema in association 16 with hypoxemia and respiratory acidosis, metabolic acidosis, shift of the oxyhemoglobin dissociation 17 curve to the left, and arterial hypotension ( Douglas et al., 1989). A characteristic of NO 2 intoxication 18 after the acute phase is a period of apparent recovery followed by late onset bronchiolar injury that 19 manifests as bronchiolitis fibrosa obliterans ( NIOSH, 1976; NRC, 1977; Hamilton, 1983; Douglas 20 et al., 1989). 21 22 Toxicity from acute exposure can be described in one of three categories: 1) immediate death 23 after very heavy exposure, 2) delayed symptoms with development of edema within 48 hours, and 24 3) apparent recovery from immediate effects but later chronic chest disease of varying severity ( NRC, 25 1977; Hamilton, 1983). Morphological and biochemical changes in the lungs during these phases 26 were studied in mice exposed to 140 ppm for 1 hour ( Siegel et al., 1989). Immediately after 27 exposure, cell death was noted in areas adjacent to the distal terminal bronchioles, and protease 28 inhibitor activity, lung protein content, and lung wet weight were significantly elevated. Two days 29 after exposure, the histological damage was exacerbated with complete obliteration of the alvoelar 30 structure, progressive edema and congestion of the lungs, hypertrophy and hyperplasia of the 31 epithelial cells, increased numbers of intraalveolar macrophages and neutrophils. Also two days after 32 exposure, there were dose related increases in   glucuronidase, lactate dehydrogenase, and choline 33 kinase activities as well as increased protease inhibitor activity, pulmonary protein, and lung wet 34 weight. Pulmonary injury is characterized by loss of ciliated cells, disruption of tight capillary 35 junctions, degeneration of type I cells, and proliferation of type II cells ( Siegel et al., 1989; Elsayed, 36 1994). 37 38 The predominant reaction in the lungs involves hydrogen abstraction by readily oxidizable 39 tissue components such as proteins and lipids to form nitrous acid and the nitrite radical ( Postlethwait 40 and Bidani, 1994; U. S. EPA, 1995) and reaction with water to form nitrous and nitric acids 41 ( Greenbaum et al., 1967; Goldstein et al., 1977). This reaction can lead to one mechanism by which 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 34 NO 2 causes pulmonary injury, lipid peroxidation. NO 2 is a free radical that can attack unsaturated 1 fatty acids in the cell membrane forming carbon and oxygen centered radicals in a chain reaction 2 ( Ainslie, 1993; Elsayed, 1994; U. S. EPA, 1995). This theory is supported by studies on the effects 3 of antioxidants on NO 2 exposure in humans and animals. Four week supplementation with vitamins 4 C and E before exposure to 4 ppm for 3 hours resulted in a marked decrease in the amount of 5 conjugated dienes and attenuated the decrease in elastase inhibitory capacity in the alveolar lining 6 fluid of healthy, human volunteers ( Mohsenin, 1991). Guinea pigs maintained on an ascorbic acid 7 deficient diet had increased lung lavage fluid protein following exposure to 4.8 ppm NO 2 for 3 hours 8 and increased wet lung weight, increased nonprotein sulfhydryl and ascorbic acid content of the lungs, 9 and decreased   tocopherol content of the lungs following exposure to 4.5 ppm for 16 hours. These 10 changes were not seen in animals maintained on normal guinea pig diets ( Hatch et al., 1986). Rats 11 exposed to 30 and 40 ppm for 4 hours had elevations of lactate dehydrogenase ( LDH), malate 12 dehydrogenase ( MDH), and glucose 6 phosphate dehydrogenase ( GDH), in lavage fluid which were 13 significantly attenuated in animals maintained on diets providing 1000 mg/ kg of   tocopherol ( Guth 14 and Mavis, 1986). Another study found changes in fatty acid composition of alveolar lavage 15 phospholipids following exposure of rats to 10 ppm NO 2 for 12 hours ( Kobayashi et al., 1984). 16 17 4.3. Oxides of Nitrogen 18 19 NO 2 exists as an equilibrium mixture of NO 2 and N 2 O 4 but the dimer is not important at 20 ambient concentrations ( U. S. EPA, 1993). The two compounds are phase related forms with N 2 O 4 21 favored in the liquid phase and NO 2 favored in the gaseous phase. An equilibrium distribution is 22 reached which favors the lowest energy state in the phase. As a result when N 2 O 4 is released it 23 vaporizes and dissociates into NO 2, making it nearly impossible to generate a significant concentration 24 of N 2 O 4 at atmospheric pressure and ambient temperatures, without generating a vastly higher 25 concentration of NO 2. Because of this effect, almost no inhalation toxicity data are available on N 2 O 4. 26 27 Another oxide of nitrogen, nitric oxide ( NO), is unstable in air and undergoes spontaneous 28 oxidation to NO 2. If the exposure concentration of NO is not high enough to be lethal due to 29 methemoglobin formation, the victim can recover completely. On the other hand, concentrations of 30 NO 2 that are not rapidly lethal may cause more persistent effects and in some cases cause death from 31 pulmonary edema after a delay of several days ( NIOSH, 1976). In photochemical smog, NO 2 absorbs 32 sunlight of wavelengths between 290 430 nm and decomposes to NO and O ( U. S. EPA, 1993). If 33 NO is of concern, reference should be made to the AEGL technical support document for nitric 34 oxide. 35 36 4.4. Other Relevant Information 37 4.4.1. Species Variability 38 39 Several studies indicate that there is a size dependent species sensitivity to NO 2 with larger 40 animals apparently less sensitive than smaller animals such as rodents. Dogs showed only mild signs 41 of irritation at concentrations that caused pulmonary edema in rats ( Carson et al., 1962). Dogs also 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 35 survived exposures to 1000 ppm for 136 minutes and 5000 ppm for up to 22 minutes ( Greenbaum 1 et al., 1967) and sheep survived exposures to 500 ppm for 15 20 minutes ( Januszkiewicz and 2 Mayorga, 1994). In contrast, 15 minute and 1 hour LC 50 values in the rat ranged from 201 420 ppm 3 and 115 168 ppm, respectively ( Gray et al., 1954; Carson et al., 1962). Based on the data available, 4 humans are not more sensitive than larger laboratory animals. For example, irritation was reported 5 for humans exposed to 30 ppm for 2 hours ( Henschler et al., 1960), in dogs exposed to 20 ppm for 6 24 hours ( Hine et al., 1970), and in monkeys exposed to 35 ppm for 2 hours ( Henry et al., 1969). 7 8 Elsayed et al. ( 2002) examined species variability through dosimetry; the calculated total 9 inspired dose from experimental measurements in rats and sheep was compared to the theoretical 10 dose of an average human. Whether normalized for body weight, lung volume, or alveolar surface 11 area, the total dose was rats >> sheep > humans. One explanation for these differences is that, upon 12 NO 2 exposure, rats exhibit an immediate decrease in minute ventilation due to a decrease in tidal 13 volume with no change in respiratory rate while the larger animals show an increase in minute 14 ventilation due to increased respiratory rate. Taking physiologic and anatomical factors into 15 consideration, rats had a much higher effective dose than the larger animals ( Elsayed et al., 2002). 16 17 4.4.2. Susceptible Populations 18 19 For chronic, low level exposures, U. S. EPA ( 1995) has identified two populations as 20 potentially at risk from NO 2 exposure: children ages 5 12 and persons with pre existing respiratory 21 disease. Conclusions drawn from epidemiology studies were that children ages 5 12 years old had 22 an increased risk of about 20% for developing respiratory symptoms and disease with each increase 23 of 0.015 ppm in estimated 2 week average NO 2 exposure ( mean weekly concentrations in bedrooms 24 0.008 0.065 ppm) and that no evidence for increased risk was found for infants < 2 years old. This 25 conclusion is supported somewhat by animal data in which adult animals were more sensitive than 26 neonates to the effects of NO 2 ( Azoulay Dupuis et al., 1983; Stephens et al., 1978). Reduced 27 ventilatory reserves may prevent individuals with respiratory disease from resuming normal activity 28 following exposure to NO 2 ( U. S. EPA, 1995). However, it is not certain whether these populations 29 are also at particular risk from acute exposure scenarios. 30 31 Taken together, the data summarized in section 2.2.3 indicate that some asthmatics exposed 32 to 0.3 0.5 ppm NO 2 may respond with either subjective symptoms or slight changes in pulmonary 33 function of no clinical significance. Also at approximately these same concentrations of NO 2 some 34 asthmatics may show slight hyperreactivity to a bronchial challenge, but the response is not more 35 severe than to NO 2 alone ( e. g. while some asthmatics respond to a bronchial challenge and to NO 2, 36 the response to the challenge is not additively increased from prior exposure to NO 2). In contrast, 37 some asthmatics did not respond to NO 2 with changes in pulmonary function or symptoms at 38 concentrations of 0.5 4 ppm. The responses of healthy individuals to NO 2 exposures are also variable 39 with some, but not all, having slight changes in pulmonary function following exposure to 5 ppm. 40 All reported responses in both asthmatic and healthy subjects at the concentrations discussed were 41 slight and of questionable biological or clinical significance. 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 36 Conclusions regarding differences in susceptibility between healthy and asthmatic individuals 1 are difficult to draw from the available data because of the high variability in responses among both 2 classifications of individuals. There is only one study which has measured the responses of both 3 healthy and asthmatic individuals with the same study protocol and experimental setup ( Linn and 4 Hackney, 1983). Dose response patterns are not discernable at these low concentrations and clear 5 thresholds are not apparent. Some individuals reported clinical symptoms in the absence of changes 6 in pulmonary function, while other individuals had measurable changes in pulmonary function tests 7 but no symptoms. One proposed explanation for the variability in the responses of asthmatics to 8 inhaled NO 2 is the existence of a subgroup of " responders." From one laboratory, several asthmatics 9 were identified as equally responsive to 0.3 ppm in more than one study ( Bauer et al., 1985; 1986). 10 However, the investigators could find no common identifiers for these " responders" such as degree 11 of baseline obstruction or their inherent airway reactivity to carbachol or cold air ( Utell, 1989). 12 Although some individuals may respond at concentrations below which others might show a 13 measurable response, the magnitude of the reported changes was not biologically or clinically 14 significant in either asthmatics or healthy individuals. 15 16 4.4.3. Concentration Response Relationship 17 18 As discussed below for AEGL 2 and  3 levels, extrapolations were made to each of the time 19 points using Cn × t = k where n = 3.5 ( ten Berge et al., 1986). The value of n was calculated by ten 20 Berge et al. based on the data of Hine et al. ( 1970). The large value of n indicates that concentration 21 is more important than duration for the effects of exposure to NO 2. 22 23 4.4.4. Susceptibility to infection 24 25 To determine the effects of NO 2 on resistance to infection, squirrel monkeys were challenged 26 with Klebsiella pneumoniae within 24 hour after exposure. No deaths occurred from exposure to 27 NO 2 alone, however, 3/ 3 monkeys died within 72 hours after 50 ppm NO 2 exposure for 2 hours 28 followed by K. pneumoniae challenge; massive infection was present in the lungs and other organs. 29 Exposure of monkeys to 10 ppm for 2 hours followed by K. pneumoniae challenge 3 5 days later did 30 not result in death of the animals, but bacteria were still present in lung tissue at necropsy up to 46 31 days after challenge indicating reduced clearence ( Henry et al., 1969). 32 33 Numerous studies have reported enhanced susceptibility of mice to infectious agents following 34 exposure to NO 2. Most of these studies have been reviewed by U. S. EPA ( 1993) and only a few are 35 described here. A single 3 hour exposure to 2.0 or 3.5 ppm NO 2 enhanced the susceptibility of three 36 strains of mice to streptococcal pneumonia and influenza infection as seen by excess mortality and 37 reduced survival time ( Ehrlich, 1978). Pulmonary bacterial defenses against Staphylococcus aureus 38 were suppressed following exposure of Swiss mice to concentrations of

4 ppm for 4 hours ( Jakab, 39 1987). Significantly decreased pulmonary bactericidal activity was shown in Swiss mice infected with 40 S. aureus then exposed to 7, 9.2, and 14.8 ppm NO 2 for 4 hours, or exposed to 2.3 and 6.6 ppm for 41 17 hours prior to infection. Histologically the lungs of mice exposed to

9.2 ppm for 4 hours showed 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 37 vascular hyperemia while those from mice exposed to

2.3 ppm for 17 hours had minor vascular 1 hyperemia and interstitial edema ( Goldstein et al., 1973). Enhanced susceptibility to infection was 2 observed in CD 1 mice exposed to 5 ppm for 6 hours/ day on two consecutive days prior to 3 inoculation with murine cytomegalovirus, followed by exposure to 5 ppm NO 2 for 6 hours/ day for 4 four consecutive days; there was no histological evidence of lung injury ( Rose et al., 1989). 5 Continuous exposure of mice to 20 ppm NO 2 for 4 days resulted in impairment of acquired resistance 6 ( decreased ED 50) of C57Bl/ 6 mice immunized prior to Klebsiella pneumoniae challenge ( Bouley et 7 al., 1986). 8 9 Mice have also been used extensively as a model for immune function alterations following 10 NO 2 exposure. Decreases in splenic and thymic weights, cellularity, plaque forming cell ( PFC) 11 responses, and hemagglutinins ( HA), along with decreased body weights, were observed in C56Bl/ 6 12 mice exposed to 20 ppm NO 2 for 48 hours ( Azoulay Dupuis et al., 1985). Significant suppression 13 of primary antibody responses ( HA and PFC) were also seen following exposure of BALB/ c mice to 14 20 and 40 ppm for 12 hours ( Hidekazu and Fujio, 1981). Other effects of NO 2 on cellular and 15 humoral immunity have been reviewed by U. S. EPA ( 1993) but are not relevant to derivation of 16 AEGL values. 17 18 19 5. DATA ANALYSIS FOR AEGL 1 20 21 AEGL 1 is the airborne concentration ( expressed as parts per million or milligrams per cubic 22 meter [ ppm or mg/ m3]) of a substance above which it is predicted that the general population, 23 including susceptible individuals, could experience notable discomfort, irritation, or certain 24 asymptomatic, non sensory effects. However, the effects are not disabling and are transient and 25 reversible upon cessation of exposure. 26 27 5.1. Summary of Human Data Relevant to AEGL 1 28 29 The evidence indicates that some asthmatics exposed to 0.3 0.5 ppm NO 2 may respond with 30 either subjective symptoms or slight changes in pulmonary function of no clinical significance. Also 31 at approximately these same concentrations of NO 2 some asthmatics may show slight hyperreactivity 32 to a bronchial challenge, but the response is not more severe than to NO 2 alone ( e. g. while some 33 asthmatics respond to a bronchial challenge and to NO 2, the response to the challenge is not additively 34 increased from prior exposure to NO 2). In contrast, some asthmatics did not respond to NO 2 at 35 concentrations of 0.5 4 ppm. The responses of healthy individuals to NO 2 exposures are also variable 36 with some, but not all, responding to 5 ppm. 37 38 Kerr et al. ( 1978; 1979) reported that 7/ 13 asthmatics experienced slight burning of the eyes, 39 slight headache, chest tightness, or labored breathing with exercise during exposure to 0.5 ppm for 40 2 hours; at this concentration the odor of NO 2 was perceptible but the subjects became unaware of 41 it after about 15 minutes. No changes in any pulmonary function tests were found immediately 42 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 38 following the chamber exposure ( Kerr et al., 1978; 1979). Significant group mean reductions in FEV 1 1 ( 17.3% vs  10.0%) and specific airway conductance ( 13.5% vs  8.5%) occurred in asthmatics after 2 exercise during exposure to 0.3 ppm for 4 hours and 1/ 6 individuals experienced chest tightness and 3 wheezing ( Bauer et al., 1985). The onset of effects was delayed when exposures were by oral nasal 4 inhalation compared with oral inhalation. This delay may result from scrubbing within the upper 5 airway. In a similar study, asthmatics exposed to 0.3 ppm for 30 minutes at rest followed by 10 6 minutes of exercise had significantly greater reductions in FEV 1 ( 10% vs 4% with air) and partial 7 expiratory flow rates at 60% of total lung capacity, but no symptoms were reported ( Bauer et al., 8 1986). In a preliminary study with 13 asthmatics exposed to 0.3 ppm for 110 minutes, slight cough 9 and dry mouth and throat and significantly greater reduction ( 11% vs 7%) in FEV 1 occurred after 10 exercise, however, in a larger study, no changes in pulmonary function were measured and no 11 symptoms were reported following exposure of 21 asthmatics to concentrations up to 0.6 ppm for 12 75 minutes ( Roger et al., 1990). The mean drop in FEV 1 for asthmatics during a 3 hour exposure 13 with intermittent exercise to 1 ppm NO 2 ( 2.5%) was significantly greater than the drop during air 14 ( 1.3%) exposure with intermittent exercise; in BALF, levels of 6 keto prostaglandin 1  were decreased 15 and levels of thromboxane B 2 and prostaglandin D 2 were increased after NO 2 exposure ( Jörres et al., 16 1995). 17 18 5.2. Summary of Animal Data Relevant to AEGL 1 19 20 Animal data relevant to derivation of AEGL 1 are limited. Slight irritation was noted in 21 squirrel monkeys exposed to 10 and 15 ppm for 2 hours ( Henry et al., 1969) and mild sensory effects 22 occurred in dogs exposed to 125 ppm for 5 minutes, 52 ppm for 15 minutes, or 39 ppm for 60 23 minutes ( Carson et al., 1962). 24 25 5.3. Derivation of AEGL 1 26 27 The study by Kerr et al., ( 1978; 1979) was considered the most appropriate to use as the basis 28 for AEGL 1 values. Exposure of asthmatics to 0.5 ppm for 2 hours resulted in clinical signs but no 29 changes in pulmonary function. Therefore, a concentration of 0.5 ppm was adopted for all time 30 points ( Table 4). The NAC agreed to the same value for all time points for AEGL 1 because it was 31 believed that these effects would appear immediately and would not progress in severity with 32 continued exposure. In addition, animal responses to NO 2 exposure have demonstrated a much 33 greater dependence upon concentration than upon time; therefore, extending the 2 hour concentration 34 to 8 hours should not exacerbate the human response. 35 36 TABLE 4. AEGL 1 Values for Nitrogen Dioxide ( ppm [ mg/ m3]) 37 10 minute 38 30 minute 1 hour 4 hour 8 hour 0.50 [ 0.94] 39 0.50 [ 0.94] 0.50 [ 0.94] 0.50 [ 0.94] 0.50 [ 0.94]

40 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 39 1 6. DATA ANALYSIS FOR AEGL 2 2 3 AEGL 2 is the airborne concentration ( expressed as ppm or mg/ m3) of a substance above 4 which it is predicted that the general population, including susceptible individuals, could experience 5 irreversible or other serious, long lasting adverse health effects or an impaired ability to escape. 6 7 6.1. Summary of Human Data Relevant to AEGL 2 8 9 Human data relevant to AEGL 2 are limited but consistent. Henschler et al. ( 1960) performed 10 several experiments on healthy, male volunteers and found that exposure to 30 ppm for 2 hours 11 caused definite discomfort. Three individuals exposed to 30 ppm for 2 hours perceived an intense 12 odor upon entering the chamber which quickly diminished and was completely absent after 25 40 13 minutes. One individual experienced a slight tickling of the nose and throat mucous membranes after 14 30 minutes, the two others after 40 minutes. From 70 minutes on, all subjects experienced a burning 15 sensation and an increasingly severe cough for the next 10 20 minutes, but coughing decreased from 16 100 minutes on. However, the burning sensation continued and moved into the lower sections of the 17 airways and was finally felt deep in the chest. At this time, marked sputum secretion and dyspnea was 18 noted. Towards the end of the exposure, the subjects' condition was described as bothersome and 19 barely tolerable. A sensation of pressure and increased sputum secretion continued for several hours 20 after cessation of exposure ( Henschler et al., 1960). In a similar experiment ( Henschler and Lütke, 21 1963) groups of 4 or 8 healthy, male volunteers were exposed to 10 ppm for 6 hours or to 20 ppm 22 for 2 hours. All subjects upon entering the chamber noted the odor which diminished rapidly. At 20 23 ppm minor scratchiness of the throat was felt after about 50 minutes and 3/ 8 experienced slight 24 headaches towards the end of the exposure period. 25 26 6.2. Summary of Animal Data Relevant to AEGL 2 27 28 Several animal studies are relevant to AEGL 2 derivation. Hine et al. ( 1970) noted 29 lacrimation, reddening of the conjunctivae, and increased respiration in 5 species exposed to

40 ppm 30 for varying durations. Lethality did not occur until concentrations and durations reached 75 ppm for 31 4 hours in the dog and 1 hour in the rabbit, 50 ppm for 1 hour in the guinea pig, and 50 ppm for 24 32 hours in the rat and mouse. At 20 ppm for 24 hours, all species showed minimal signs of irritation 33 and changes in behavior with histopathological lesions described as questionable evidence of lung 34 congestion and interstitial inflammation. 35 36 Exposure of monkeys to 35 ppm for two hours resulted in irritation as measured by changes 37 in lung function and microscopic lesions in the lung ( Henry et al., 1969). The histological lesions in 38 the lung were characterized by Siegel et al. ( 1989) following exposure of mice to 140 ppm for 1 hour. 39 Carson et al. ( 1962) conducted a series of experiments in dogs and rats. Mild irritation and some 40 respiratory effects, but no gross or microscopic lesions, were noted in dogs exposed to 53 or 39 ppm 41 NITROGEN DIOXIDE NAC/ PROPOSED 1: 01/ 2003 40 for 1 hour while rats exposed to 72 ppm for 1 hour showed signs of severe respiratory distress and 1 eye irritation as well as gross lesions in the lung and evidence of infection. 2 3 Developmental delays and disturbances in neuromotor development were reported for rat 4 pups following maternal exposure ( Tabacova et al., 1985). However, these effects were reported to 5 have occurred at levels near ambient concentrations and are well below those of most other studies 6 in both humans and animals. 7 8 6.3. Derivation of AEGL 2 9 10 From both human and animal data, it appears that a concentration of

30 ppm NO 2 is required 11 before marked irritation, discomfort, and respiratory effects occur. Therefore, a concentration of 30 12 ppm for a 2 hour exposure of humans ( Henschler et al., 1960) was used to derive AEGL 2 values. 13 Values scaled for the derivation of the 10 and 30 minute and 1, 4, and 8 hour AEGL 2 endpoints 14 were calculated from Cn × t = k using n = 3.5 ( ten Berge et al., 1986). The value of n was calculated 15 by ten Berge et al. from the data of all species together from Hine et al. ( 1970). An uncertainty factor 16 of 3 was applied to account for sensitive subpopulations. It is felt that additional uncertainty factors 17 are unnecessary because the experimental data at lower concentrations in asthmatics and persons with 18 respiratory disease are inconclusive and the effects are of questionable biological significance. 19 Furthermore, with additional uncertainty factors the values would be inconsistent with some of the 20 clinical data for asthmatics, e. g., the no adverse effect concentration of 4 ppm in the study by Linn 21 and Hackney ( 1984). Proposed AEGL 2 values are presented in Table 5. 22 23 24 TABLE 5: AEGL 2 Values for Nitrogen Dioxide ( ppm [ mg/ m3]) 25 10 minute 26 30 minute 1 hour 4 hour 8 hour 20 [ 38] 27 15 [ 28] 12 [ 23] 8.2 [ 15] 6.7 [ 13]