### DRAFT - DO NOT CITE OR QUOTE EPA/635/R-11/013C www.epa.gov/iris



# TOXICOLOGICAL REVIEW

OF

AMMONIA

(CAS No. 7664-41-7)



# In Support of Summary Information on the Integrated Risk Information System (IRIS)

October 2011

NOTICE

This document is an Interagency Science Consultation draft . This information is distributed solely for the purpose of pre-dissemination peer re view under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency determination or policy. It is being circulated for review of its technical accuracy and science policy implic ations.

U.S. Environmental Protection Agency

Washington, DC

### ii DRAFT - DO NOT CITE OR QUOTE DISCLAIMER

This document is a preliminary draft for review pu rposes only. This information is distributed solely for the purpose of pre-dissemina tion peer review under applicable information quality guidelines. It has not been formally disse minated by EPA. It does not represent and should not be construed to represent any Agency det ermination or policy. Mention of trade names or commercial products does not constitute en dorsement or recommendation for use.

### iii DRAFT - DO NOT CITE OR QUOTE

CONTENTS—TOXICOLOGICAL REVIEW OF AMMONIA (CAS No. 7664-41-7)

LIST OF TABLES ....................................

................................................... ....................................v LIST OF FIGURES ...................................

................................................... ................................. vi LIST OF ABBREVIATIONS AND ACRONYMS ................

................................................... .. vii FOREWORD ..........................................

................................................... .................................... ix AUTHORS, CONTRIBUTORS, AND REVIEWERS ..............

................................................... ..x

1. INTRODUCTION ..................................

................................................... .................................1

2. CHEMICAL AND PHYSICAL INFORMATION .............

................................................... ....3

3. TOXICOKINETICS ................................

................................................... ................................5 3.1. ABSORPTION ..................................

................................................... ............................5 3.1.1. Inhalation Exposure .......................

................................................... .....................5 3.1.2. Oral Exposure .............................

................................................... ........................6 3.1.3. Dermal Exposure ...........................

................................................... .....................6 3.2. DISTRIBUTION ................................

................................................... ...........................7 3.2.1. Inhalation Exposure .......................

................................................... .....................8 3.2.2. Oral Exposure .............................

................................................... ........................8 3.2.3. Dermal Exposure ...........................

................................................... .....................8 3.3. METABOLISM ..................................

................................................... ...........................8 3.4. ELIMINATION .................................

................................................... ..........................11 3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS ............................13

4. HAZARD IDENTIFICATION .........................

................................................... .....................14 4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL CONTROLS ..........................................

................................................... ......................15 4.1.1. Case Reports – Oral and Inhalation Exposure s ................................................. ..15 4.1.2. Controlled Human Inhalation Exposure Studie s ................................................. 15 4.1.3. Cross Sectional Studies in Farmers Exposed to Inhaled Ammonia.....................18 4.1.4. Occupational Studies in Industrial Worker P opulations ......................................19 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIO ASSAYS IN ANIMALS—ORAL AND INHALATION ....................... .............................................27 4.2.1. Oral Exposure .............................

................................................... ......................27 4.2.2. Inhalation Exposure .......................

................................................... ...................31 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION ........................................

................................................... .....................38 4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES ....................................39 4.4.1. Acute Oral Studies ........................

................................................... ....................39 4.4.2. Acute and Short-Term Inhalation Studies ...

................................................... .....39 4.4.3. Immunotoxicity ............................

................................................... .....................40 4.5. MECHANISTIC STUDIES .........................

................................................... ................46 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS ........ ...........................................46 4.6.1. Oral ......................................

................................................... .............................46 4.6.2. Inhalation ................................

................................................... ..........................47 4.6.3. Mode-of-Action Information ................

................................................... ............51 4.7. EVALUATION OF CARCINOGENICITY ...............

................................................... 51 4.7.1. Summary of Overall Weight-of-Evidence .....

................................................... ...51 4.7.2. Synthesis of Human, Animal, and Other Suppo rting Evidence ..........................51

### iv DRAFT - DO NOT CITE OR QUOTE

4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES ..... ..........................................51 4.8.1. Possible Childhood Susceptibility .........

................................................... ...........51 4.8.2. Possible Gender Differences ...............

................................................... ..............52 4.8.3. Other Susceptible Populations .............

................................................... .............52

5. DOSE-RESPONSE ASSESSMENTS .....................

................................................... ..............53 5.1. ORAL REFERENCE DOSE (RfD) ...................

................................................... ..........53 5.1.1. Previous RfD Assessment ...................

................................................... ..............55 5.2. INHALATION REFERENCE CONCENTRATION (RfC) .... ......................................55 5.2.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification .....................................

................................................... .................55 5.2.2. Methods of Analysis .......................

................................................... ..................60 5.2.3. RfC Derivation—Including Application of Unc ertainty Factors (UFs) ..............60 5.2.4. Previous RfC Assessment ...................

................................................... ..............62 5.3. CANCER ASSESSMENT ...........................

................................................... ................63

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HA ZARD AND DOSE RESPONSE...........................................

................................................... ................................64 6.1. HUMAN HAZARD POTENTIAL ......................

................................................... ........64 6.2. DOSE RESPONSE ...............................

................................................... .......................65 6.2.1. Noncancer/Oral ............................

................................................... .....................65 6.2.2. Noncancer/Inhalation ......................

................................................... ..................65 6.2.3. Cancer ....................................

................................................... ...........................66

7. REFERENCES ....................................

................................................... ..................................67

APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PU BLIC COMMENTS AND DISPOSITION ...................................

................................................... ...................... A-1

APPENDIX B. SUMMARY OF REPEAT DOSE TOXICITY INFORM ATION FOR SELECTED AMMONIUM SALTS ...........................

................................................... ........B-1

APPENDIX C. SUPPLEMENTAL INFORMATION ON AMMONIA .. .................................C-1 C.1. AMMONIA LEVELS MEASURED IN EXPIRED AIR IN HUM ANS ....................C-1 C.2. HUMAN CASE STUDIES AND REPORTS OF HUMAN EXPOS URE TO AMMONIA............................................

................................................... ....................C-8 C.3. CONTROLLED HUMAN EXPOSURE STUDIES OF AMMONIA INHALATION ........................................

................................................... .................C-33 C.4. CROSS SECTIONAL STUDIES OF LIVESTOCK FARMERS EXPOSED TO AMMONIA............................................

................................................... ..................C-36 C.5. ACUTE AND SHORT-TERM INHALATION TOXICITY STUD IES OF AMMONIA IN EXPERIMENTAL ANIMALS ................... .....................................C-40 C.6. MECHANISTIC STUDIES .........................

................................................... ...........C-48

### v DRAFT - DO NOT CITE OR QUOTE LIST OF TABLES

Table 2-1. Chemical and physical properties of amm onia .............................................. .............. 3

Table 4-1. Symptoms and lung function results of w orkers exposed to different levels of TWA ammonia concentrations ............................

................................................... .......................... 21

Table 4-2. The prevalence of respiratory symptoms and disease in urea fertilizer workers exposed to ammonia ................................

................................................... ............................ 22

Table 4-3. Logistic regression analysis of the rel ationship between ammonia concentration and respiratory symptoms or disease in exposed urea fer tilizer workers ................................... .. 22

Table 4-4. Prevalence of acute respiratory symptom s and cross-shift changes in lung function among workers exposed to ammonia in a urea fertiliz er factory ........................................ ... 25

Table 4-5. Summary of significant changes in serum from workers occupationally exposed to ammonia at a fertilizer plant......................

................................................... .......................... 26

Table 4-6. Effect of ammonia in drinking water on t he thickness of the gastric antral and body mucosa of the rat stomach .........................

................................................... .......................... 27

Table 4-7. Effect of ammonia in drinking water on g astric antral and body mucosa in the stomach of Sprague-Dawley rats administered 0.01% a mmonia in drinking water .............. 28

Table 4-8. Summary of histological changes observe d in rats exposed to ammonia for 6 weeks34

Table 4-9. Incidence of pulmonary lesions in rats inoculated with M. pulmonis and exposed to ammonia (7 days later for 28–42 days) .............

................................................... .................. 41

Table 4-10. Mortality in P. multocida -infected mice exposed to ammonia for 8 or 168 hour s .. 42

Table 4-11. Dermal response to the injection of tu berculin in animals exposed to ammonia for 3 weeks (mean diameter of redness in mm) ............

................................................... ............... 44

Table 4-12. Summary of noncancer results of human occupational studies and repeat-dose studies in experimental animals involving inhalatio n exposure to ammonia......................... 49

Table B-1. Summary of noncancer results of repeat dose studies of oral exposure of experimental animals to selected ammonium salts ...

................................................... ........ B-1

Table C-1. Ammonia levels in exhaled breath of vol unteers............................................ ......... C-2

Table C-2. Human case studies and reports of human exposure to ammonia ......................... C-10

Table C-3. Controlled human exposure studies of am monia inhalation .................................. C-33

Table C-4. Cross sectional studies of livestock fa rmers exposed to ammonia ........................ C -36

Table C-5. Acute and short-term inhalation toxicit y studies of ammonia in animals ............. C-41

Table C-6. In vivo genotoxicity studies of ammonia

.................................................. ............. C-51

Table C-7. In vitro genotoxicity studies of ammoni a ................................................. ............. C-52

### vi DRAFT - DO NOT CITE OR QUOTE LIST OF FIGURES

Figure 3-1. Glutamine cycle in the liver. ........

................................................... ............................ 9

Figure 3-2. The urea cycle showing the compartment alization of its steps within liver cells. .... 10

Figure 5-1. Exposure-response array comparing work ers occupationally exposed and human volunteers acutely exposed to ammonia..............

................................................... ................ 57

Figure 5-2. Exposure-response array comparing nonc ancer effects in occupationally exposed workers and experimental animals exposed to ammonia by inhalation. ................................ 59

### vii DRAFT - DO NOT CITE OR QUOTE LIST OF ABBREVIATIONS AND ACRONYMS

ACGIH American Conference of Governmental Industrial Hygi enists ALP alkaline phosphatase ALT alanine aminotransferase AMP adenosine monophosphate AST aspartate aminotransferase ATSDR Agency for Toxic Substances and Disease Registry ATSG acid-treated silica gel BMD benchmark dose BrDU 5-bromo-2-deoxyuridine BUN blood urea nitrogen C3 complement 3 CAC cumulative ammonia concentration CASRN Chemical Abstracts Service Registry Number CI confidence interval FEF forced expiratory flow FEV forced expiratory volume FVC forced vital capacity GABA γ -amino butyric acid IgE immunoglobin E IgG immunoglobin G IRIS Integrated Risk Information System LOAEL lowest-observed-adverse-effect level MAO monoamine oxidase MMEF mean midexpiratory flow MNNG N-methyl-N’-nitro-N-nitrosoguanidine MRM murine respiratory mycoplasmosis NAD + 

### nicotineamide adenine dinucleotide, oxidized NADH nicotineamide adenine dinucleotide, reduced NH 3 

### ammonia NH 4 + 

### ammonium ion NIOSH National Institute for Occupational Safety and Heal th NMDA N-methyl D-aspartate NOAEL no-observed-adverse-effect level NOx nitrogen oxides NRC National Research Council OR odds ratio PARP poly(ADP-ribose) polymerase PEF peak expiratory flow PEFR peak expiratory flow rate PHA phytohemagglutin POD point of departure PPD purified protein derivative PRRSV porcine reproductive and respiratory syndrome virus RfC reference concentration RfD reference dose SIFT-MS selected ion flow tube mass spectrometry

### viii DRAFT - DO NOT CITE OR QUOTE

TLV threshold limit value TWA time-weighted average UF uncertainty factor U.S. EPA U.S. Environmental Protection Agency

### ix DRAFT - DO NOT CITE OR QUOTE FOREWORD

The purpose of this Toxicological Review is to prov ide scientific support and rationale for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to ammonia. It is not intended to be a comprehensive treatise o n the chemical or toxicological nature of

ammonia.
The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose
Response, is to present the major conclusions reached in the derivation of the reference dose,
reference concentration, and cancer assessment, whe re applicable, and to characterize the overall confidence in the quantitative and qualitative aspe cts of hazard and dose response by addressing the quality of data and related uncertainties. The discussion is intended to convey the limitations of the assessment and to aid and guide the risk ass essor in the ensuing steps of the risk

assessment process. For other general information about this assessment or other questions relating to IRIS, the reader is referred to EPA’s IRIS Hotline at (20 2) 566-1676 (phone), (202) 566-1749 (fax), or hotline.iris@epa.gov (email address).

### x DRAFT - DO NOT CITE OR QUOTE AUTHORS, CONTRIBUTORS, AND REVIEWERS

CHEMICAL MANAGER/ AUTHOR

Audrey Galizia, Dr. PH Office of Research and Development U.S. Environmental Protection Agency Edison, NJ

CONTRIBUTORS
James Ball, Ph.D. National Center for Environmental Assessment Office of Research and Development Washington, DC

Christopher Brinkerhoff, Ph.D. National Center for Environmental Assessment Office of Research and Development Washington, DC

Christopher Sheth, Ph.D. National Center for Environmental Assessment Office of Research and Development Washington, DC

John Whalan National Center for Environmental Assessment Office of Research and Development Washington, DC

CONTRACTOR SUPPORT

Amber Bacom Fernando Llados Julie Stickney Chemical, Biological and Environmental Center SRC, Inc.

Portions of this Toxicological Review were develop ed under a Memorandum of Understanding with the Agency for Toxic Substances and Disease Registry (ATSDR) and were adapted from the Toxicological Profile for Ammonia (ATSDR, 2004) as part of a collaborative effort in the development of human health toxicolog ical assessments for the purposes of making

more efficient use of available resources and to sh are scientific information.

### xi DRAFT - DO NOT CITE OR QUOTE

REVIEWERS

INTERNAL EPA REVIEWERS

Ted Berner, M.S. National Center for Environmental Assessment Office of Research and Development Research Triangle Park, NC

Amanda S. Persad, Ph.D. National Center for Environmental Assessment Office of Research and Development Research Triangle Park, NC

Paul Reinhart, Ph.D. National Center for Environmental Assessment Office of Research and Development Research Triangle Park, NC

Marian Rutigliano, MD National Center for Environmental Assessment Office of Research and Development Washington, DC

### 1 DRAFT - DO NOT CITE OR QUOTE 1. INTRODUCTION 1

2

3 

### This document presents background information and j ustification for the Integrated Risk 4 

### Information System (IRIS) Summary of the hazard and dose-response assessment of ammonia. 5 

### IRIS Summaries may include oral reference dose (RfD ) and inhalation reference concentration 6 

### (RfC) values for chronic and other exposure duratio ns, and a carcinogenicity assessment. 7 

### The RfD and RfC, if derived, provide quantitative i nformation for use in risk assessments 8 

### for health effects known or assumed to be produced through a nonlinear (presumed threshold) 9 

### mode of action. The RfD (expressed in units of mg/ kg-day) is defined as an estimate (with 10 

### uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human 11 

### population (including sensitive subgroups) that is likely to be without an appreciable risk of 12 

### deleterious effects during a lifetime. The inhalat ion RfC (expressed in units of mg/m 3 

### ) is 13 

### analogous to the oral RfD, but provides a continuou s inhalation exposure estimate. The 14



### inhalation RfC considers toxic effects for both the respiratory system (portal-of-entry) and for 15 

### effects peripheral to the respiratory system (extra respiratory or systemic effects). Reference 16 

### values are generally derived for chronic exposures (up to a lifetime), but may also be derived for 17 

### acute ( ≤ 24 hours), short-term (>24 hours up to 30 days), an d subchronic (>30 days up to 10% of 18 

### lifetime) exposure durations, all of which are deri ved based on an assumption of continuous 19 

### exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are 20 

### derived for chronic exposure duration. 21 

### The carcinogenicity assessment provides information on the carcinogenic hazard 22 

### potential of the substance in question and quantita tive estimates of risk from oral and inhalation 23 

### exposure may be derived. The information includes a weight-of-evidence judgment of the 24 

### likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic 25 

### effects may be expressed. Quantitative risk estima tes may be derived from the application of a 26 

### low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on 27 

### the estimate of risk per mg/kg-day of oral exposure . Similarly, an inhalation unit risk is a 28 

### plausible upper bound on the estimate of risk per μ g/m 3 

### air breathed. 29 

### Development of these hazard identification and dose -response assessments for ammonia 30 

### has followed the general guidelines for risk assess ment as set forth by the National Research 31 

### Council (NRC, 1983). U.S. Environmental Protection Agency (U.S. EPA) Guidelines and Risk 32 

### Assessment Forum technical panel reports that may h ave been used in the development of this 33 

### assessment include the following: Guidelines for the Health Risk Assessment of Chemic al 34



### Mixtures (U.S. EPA, 1986a), Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986b), 35 

### Recommendations for and Documentation of Biological Values for Use in Risk Assessment (U.S. 36 

### EPA, 1988), Guidelines for Developmental Toxicity Risk Assessme nt (U.S. EPA, 1991), Interim 37 

### Policy for Particle Size and Limit Concentration Is sues in Inhalation Toxicity (U.S. EPA, 38

### 2 DRAFT - DO NOT CITE OR QUOTE

1994a), Methods for Derivation of Inhalation Reference Conc entrations and Application of 1 

### Inhalation Dosimetry (U.S. EPA, 1994b), Use of the Benchmark Dose Approach in Health Risk 2



### Assessment

(U.S. EPA, 1995), Guidelines for Reproductive Toxicity Risk Assessmen t (U.S. EPA, 3 

### 1996), Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 1998), Science Policy Council 4



### Handbook : Risk Characterization (U.S. EPA, 2000a), Benchmark Dose Technical Guidance 5



### Document

(U.S. EPA, 2000b), Supplementary Guidance for Conducting Health Risk A ssessment 6 

### of Chemical Mixtures (U.S. EPA, 2000c), A Review of the Reference Dose and Reference 7



### Concentration Processes (U.S. EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. 8 

### EPA, 2005a) , Supplemental Guidance for Assessing Susceptibilit y from Early-Life Exposure to 9 

### Carcinogens (U.S. EPA, 2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 10 

### 2006a), A Framework for Assessing Health Risks of Environme ntal Exposures to Children (U.S. 11 

### EPA, 2006b), and Recommended Use of Body Weight 3/4 

### as the Default Method in Derivation of 12 

### the Oral Reference Dose (U.S. EPA, 2011a). 13 

### The literature search strategy employed for ammonia was based on the Chemical 14 

### Abstracts Service Registry Number (CASRN) and at le ast one common name. Any pertinent 15 

### scientific information submitted by the public to t he IRIS Submission Desk was also considered 16 

### in the development of this document. Primary, peer -reviewed literature identified through June 17



### 2011 was included where that literature was determi ned to be critical to the assessment. The 18 

### relevant literature included publications on ammoni a which were identified through Toxicology 19 

### Literature Online (TOXLINE), PubMed, the Toxic Subs tance Control Act Test Submission 20 

### Database (TSCATS), the Registry of Toxic Effects of Chemical Substances (RTECS), the 21 

### Chemical Carcinogenesis Research Information System (CCRIS), the Developmental and 22 

### Reproductive Toxicology/Environmental Teratology In formation Center (DART/ETIC), the 23 

### Hazardous Substances Data Bank (HSDB), the Genetic Toxicology Data Bank (GENE-TOX), 24 

### Chemical abstracts, and Current Contents. Other pe er-reviewed information, including health 25 

### assessments developed by other organizations, revie w articles, and independent analyses of the 26 

### health effects data were retrieved and may be inclu ded in the assessment where appropriate. 27

28

### 3 DRAFT - DO NOT CITE OR QUOTE 2. CHEMICAL AND PHYSICAL INFORMATION 1

2

3 

### Ammonia is a corrosive gas with a very pungent odor (O’Neil et al., 2006). It is highly 4 

### soluble in water (4.82 × 10 5 

### mg/L) and is a weak base (Lide, 2008; Eggeman, 200 1; Dean, 1985). 5 

### When ammonia (NH 3 + 

### ) is present in water at environmental pH, a pKa of 9.25 indicates that the 6 

### equilibrium will favor the formation of the conjuga te acid, the ammonium ion (NH 4 + 

### ) (Lide, 7 

### 2008). A solution of ammonia in water is sometimes referred to as ammonium hydroxide 8



### because the ammonia and water both ionize to form a mmonium cations and hydroxide anions 9 

### (Eggeman, 2001). Ammonium salts are easily dissolv ed in water and disassociate into the 10



### ammonium ion and the anion. 11 

### Many physical and chemical properties of ammonia ar e related to the pH of ammonia in 12 

### solution (ammonium hydroxide). Ammonium hydroxide is a weak base that is partially ionized 13



### in water with a dissociation constant of 1.77 x 10 -5 

### at 25 °C that increases slightly with 14 

### increasing temperature (Reed, 1982). At a pH of 8. 25, 90% of ammonia will be protonated. At a 15 

### pH of 7.25, 99% of ammonia will be protonated. Thu s, a decrease in pH would result in an 16



### increase in the ammonium ion concentration and an i ncrease in solubility of ammonia in water. 17 

### At physiological pH (7.4), the equilibrium between NH 3 

### and NH 4 + 

### favors the formation of NH 4 +



### . 18 

### Chemical and physical properties of ammonia are lis ted in Table 2-1. 19

20 

### Table 2-1. Chemical and physical properties of amm onia

Chemical name Ammonia a

Synonym(s) AM-Fol; anhydrous ammonia; ammonia gas; Nitro-sil; R 717; Spirit of hartshorn ChemID Plus, 2009

Structure



### N H

H H ChemID Plus, 2009

Chemical formula NH 3 ChemID Plus, 2009

CASRN 7664-41-7 a ChemID Plus, 2009 Molecular weight 17.031 Lide, 2008 Form Colorless gas; corrosive O’Neil et al., 2006 Melting point -77.73°C Lide, 2008 Boiling point -33.33°C Lide, 2008 Odor threshold 53 ppm O’Neil et al., 2006 Density 0.7714 g/L at 25°C O’Neil et al., 2006 Vapor density 0.5967 (air = 1) O’Neil et al., 2006 pK a (ammonium ion) 9.25 Lide, 2008 Solubility: Water Organic solvents 4.82 × 10 5 mg/L at 24°C Soluble in ethanol, chloroform, and ether Dean, 1985 Lide, 2008; O’Neil et al., 2006

### 4 DRAFT - DO NOT CITE OR QUOTE

Table 2-1. Chemical and physical properties of amm onia

Vapor pressure 7.51 × 10 3 mm Hg at 25°C AIChE, 1999 Henry’s law constant 1.61 × 10 - 5 atm-m 3 /mol at 25°C Betterton, 1992

Conversion factors ppm to mg/m 3 mg/m 3 to ppm 1 ppm = 0.707 mg/m 3 1 mg/m 3 = 1.414 ppm Verschueren, 2001 Verschueren, 2001

a Ammonia dissolved in water is sometimes referred to as ammonium hydroxide (CASRN 1336-21-6). Ammonium hydroxide does not exist outside of solution.
1 

### Ammonia is a major component of the geochemical nit rogen cycle and is essential for 2 

### many biological processes (Rosswall, 1981). Nitrog en-fixing bacteria convert atmospheric 3 

### nitrogen into ammonia available for plant uptake (S ocolow, 1999; Rosswall, 1981). Organic 4 

### nitrogen released from biota is converted into ammo nia through nitrogen mineralization 5 

### (Rosswall, 1981). Ammonia in water and soil is nat urally converted into nitrite and nitrate 6 

### through the process of nitrification (Rosswall, 198 1). 7 

### Commercially produced ammonia is obtained through t he Haber-Bosch process, which 8 

### involves mixing nitrogen from the atmosphere with h ydrogen obtained from natural gas in a 1 to 9 

### 3 ratio and passing the mixture over a catalyst at high temperature and pressure (Eggeman, 10 

### 2001). Over the past century, world-wide anthropog enic nitrogen fixation (conversion to 11 

### ammonia) has risen to approximately 140 million met ric tons, exceeding the amount of nitrogen 12 

### fixed through natural processes (NSF, 1999; Socolow , 1999). 13 

### Large amounts (thousands of tons) of ammonia are tr ansported and stored using large 14 

### pipelines and refrigerated, low pressure tanks (Egg eman, 2001). Approximately 80–85% of 15 

### commercially produced ammonia is used in the produc tion of agricultural fertilizers in the form 16 

### of urea, ammonium nitrate, ammonium sulfate, ammoni um phosphate, and other nitrogen 17 

### compounds (Eggeman, 2001). The remaining uses of a mmonia mostly involve formation of 18 

### chemical intermediates (Eggeman, 2001). Ammonia is used in metal treating operations, in 19 

### water treatment operations, in catalytic reactors, as a convenient source of hydrogen for the 20 

### hydrogenation of fats and oils, as a neutralizer in the petroleum industry, and as a stabilizer in the 21 

### rubber industry (HSDB, 2009). Ammonia may be emitt ed during these processes and may also 22 

### be emitted from light duty vehicles, heavy-duty die sel trucks and some non-road engines. 23 

### Ammonia has been used to reduce nitrogen oxides (NO x) emissions from the exhaust of 24 

### stationary combustion sources such as industrial an d municipal boilers and power generators 25 

### since the 1980’s (Johnson et al., 2009), but more r ecently, ammonia (generated from urea 26 

### injected into the exhaust stream) is being used in a selective catalytic reduction-based diesel 27 

### engine aftertreatment technology to reduce NOx emis sions. 28

### 5 DRAFT - DO NOT CITE OR QUOTE 3. TOXICOKINETICS 1 1

2

3 

### Ammonia can be absorbed by the inhalation and oral routes of exposure. There is less 4 

### certainty regarding absorption through the skin, al though absorption through the eye has been 5



### documented. Most of the inhaled ammonia is retaine d in the upper respiratory tract and is 6 

### subsequently eliminated in expired air. Ammonia th at reaches systemic circulation is widely 7 

### distributed to all body compartments, although subs tantial first-pass metabolism occurs in the 8 

### liver, where biotransformation into urea and glutam ine occur. Ammonia exists in the blood as 9 

### ammonium ion (NH 4 + 

### ). Ammonia is transported in the circulatory syste m primarily via 10 

### glutamine and alanine, amino acids that are used to transport ammonia to and from tissues. 11 

### When transported to the liver and kidney, the amide moiety is hydrolyzed via glutaminase 12 

### forming glutamatic acid (glutamate) and ammonium io n, which is synthesized into urea and 13



### excreted in the urine. Ammonia or ammonium ion rea ching the tissues is utilized for glutamate 14 

### production, which participates in transamination an d other reactions. The principal means of 15



### excretion of absorbed ammonia in mammals is as urin ary urea; minimal amounts are excreted in 16 

### the feces and in expired air. 17 

### Ammonia is endogenously produced in humans and anim als. It is an essential 18 

### mammalian metabolite used in nucleic acid and prote in synthesis, is necessary for maintaining 19 

### acid-base balance, and an integral part of nitrogen homeostasis. Given its important metabolic 20 

### role, ammonia exists in a homeostatically regulated equilibrium in the body. 21

22



### 3.1. ABSORPTION 23 

### 3.1.1. Inhalation Exposure 24 

### Experiments with volunteers 2 

### show that ammonia, regardless of its tested concen tration 25 

### in air (range, 57–500 ppm or 40–354 mg/m 3 

### ), is almost completely retained in the nasal mucos a 26 

### (83–92%) during short-term acute exposure, i.e., up to 120 seconds (Landahl and Hermann, 27 

### 1950). However, longer-term acute exposure (10–27 minutes) to a concentration of 500 ppm 28 

### (354 mg/m 3 

### ) resulted in lower retention (4–30%), with expired breath concentrations of 350–400 29 

### ppm (247–283 mg/m 3 

### ) observed by the end of the exposure period (Silve rman et al., 1949), 30 

### suggesting saturation of absorption into the nasal mucosa. Nasal and pharyngeal irritation, but 31 

### not tracheal irritation, suggests that ammonia is r etained in the upper respiratory tract. 32

1 Portions of this section were adapted from the Toxi cokinetics Section (Section 3.4) of the Toxicological Profile for Ammonia (ATSDR, 2004) under a Memorandum of Understanding (MOU) with ATSDR.

2 The human toxicokinetic studies cited in this secti on did not provide information on the human subject s research ethics procedures undertaken in the studies; howeve r, there is no evidence that the conduct of the res earch was fundamentally unethical or significantly deficient relative to the ethical standards prevailing at the time the research was conducted.

### 6 DRAFT - DO NOT CITE OR QUOTE

Unchanged levels of blood urea nitrogen (BUN), nonp rotein nitrogen, urinary urea, and urinary 1 

### ammonia following these acute exposures are evidenc e of low absorption into the blood. 2 

### Exposure to a common occupational limit of ammonia in air (25 ppm or 18 mg/m 3 

### ), assuming 3 

### 30% uptake into blood, would yield an increase in b lood ammonia concentration of 0.09 μg/mL 4 

### (calculated by WHO, 1986). This calculated rise wo uld likely be indistinguishable from the 5 

### observed baseline levels of 0.1–1.0 μg/mL (Monsen, 1987; Conn, 1972; Brown et al., 1957) for 6 

### healthy controls. 7 

### Data in rabbits and dogs provide supporting evidenc e for high-percentage nasal retention, 8 

### resulting in a lower fraction of the inhaled dose r eaching the lower respiratory tract (Egle, 1973; 9 

### Dalhamn, 1963; Boyd et al., 1944). Continuous expo sure of rats to up to 32 ppm (23 mg/m 3 

### ) for 10 

### 24 hours did not result in a statistically signific ant increase in blood ammonia levels (0.1 μg/mL 11 

### above preexposure levels), whereas exposures to 310 –1,157 ppm (219–818 mg/m 3 

### ) led to 12 

### significantly increased blood concentrations of amm onia within 8 hours of exposure initiation; 13 

### blood ammonia returned to preexposure values within 12 hours of continuous exposure 14 

### (Schaerdel et al., 1983). 15

16 

### 3.1.2. Oral Exposure 17 

### Case reports of human ingestion of household ammoni a (ammonium hydroxide) provide 18 

### evidence of oral absorption, but few quantitative d ata are available. For example, in a fatal case 19



### of a man who drank an unknown amount of a 2.4% solu tion of ammonium hydroxide, analysis of 20



### the contents of the stomach and blood showed ammoni um ion levels of 15.3 mg in the stomach 21 

### and 33 μg/mL in the blood (Klendshoj and Rejent, 19 66). This blood concentration is about 30- 22 

### fold higher than the concentration of 1 μg/mL in fa sting volunteers, as reported by Conn (1972). 23 

### Ammonium ion is endogenously produced in the human digestive tract, much of it arising 24 

### from the bacterial degradation of nitrogenous compo unds from ingested food. About 25 

### 4,200 mg/day are produced, >70% of which is synthes ized or liberated within the colon and its 26 

### fecal contents (Summerskill and Wolpert, 1970). Ab out 99% of the total amount produced 27 

### (4,150 mg) is systemically absorbed. Evidence sugg ests that fractional absorption of ammonia 28 

### increases as the lumen pH increases, and that activ e transport occurs at the lower pH levels 29 

### (absorption has been detected at a pH as low as 5) (Castell and Moore, 1971; Mossberg and 30 

### Ross, 1967). Ammonium ion absorbed from the gastro intestinal tract travels via the hepatic 31 

### portal vein directly to the liver, where in healthy individuals, most of it is converted to urea and 32 

### glutamine. 33

34 

### 3.1.3. Dermal Exposure 35 

### Quantitative data on absorption from exposure by th e dermal route are not available. One 36 

### report of five case histories of workers exposed to anhydrous ammonia via a burst gas pipe 37 

### indicated that there was systemic toxicity (vomitin g, renal congestion, and delirium), suggesting 38

### 7 DRAFT - DO NOT CITE OR QUOTE

dermal absorption; however, the fractional dose fro m dermal exposure could not be determined 1 

### (Slot, 1938). WHO (1986) concluded that systemic e ffects from skin and eye exposure are not 2 

### quantitatively important. Ammonia is readily absor bed into the eye; and it was found to diffuse 3 

### within seconds into the cornea, lens, drainage syst em, and retina (Beare et al., 1988; Jarudi and 4 

### Golden, 1973). However, amounts absorbed were not quantified, and absorption into systemic 5 

### circulation was not investigated. 6

7



### 3.2. DISTRIBUTION 8 

### The range of mean ammonia concentrations in humans as a result of endogenous 9 

### production was reported as 0.1 to 0.6 μg/mL in arte rial blood and 0.2 to 1.7 μg/mL in venous 10 

### blood (Huizenga et al., 1994). Other baseline leve ls observed in experimental volunteers range 11 

### from 1 to 5.5 μg/mL (Conn, 1972; Brown et al., 1957 ). Ammonia is homeostatically regulated to 12 

### remain at low concentrations, with 95–98% existing in the blood (at physiological pH) as NH 4 + 13 

### ion (da Fonseca-Wolhheim, 1995; Souba, 1987). 14 

### Ammonia is present in fetal circulation. In vivo s tudies in several animal species and in 15 

### vitro studies of human placenta suggest that ammoni a is produced within the uteroplacenta and 16 

### released into the fetal and maternal circulations ( Bell et al., 1989; Johnson et al., 1986; Haugel et 17 

### al., 1983; Meschia et al., 1980; Remesar et al., 19 80; Holzman et al., 1979, 1977; Rubaltelli et 18 

### al., 1968; Luschinsky, 1951). J όź wik et al. (2005) reported that ammonia levels in h uman fetal 19 

### blood (specifically, umbilical arterial and venous blood) at birth were 1.0–1.4 μg/mL, compared 20 

### to 0.5 μg/mL in the mothers’ venous blood. DeSanto et al. (1993) similarly collected human 21 

### umbilical arterial and venous blood at delivery, an d found umbilical arterial ammonia 22 

### concentrations were significantly higher than venou s concentrations; there was no correlation 23 

### between umbilical ammonia levels and gestational ag e (range of 25–43 weeks of gestation). In 24 

### sheep, uteroplacental tissues are the main site of ammonia production, with outputs of ammonia 25 

### into both the uterine and umbilical circulations (J όź wik et al., 1999). In late-gestation pregnant 26 

### sheep that were catheterized to allow measurement o f ammonia exposure to the fetus, 27



### concentrations of ammonia in umbilical arterial and venous blood and uterine arterial and venous 28 

### blood ranged from about 0.39–0.60 μg/mL (J όź wik et al., 2005, 1999). 29 

### Ammonia is present in human breast milk as one of t he sources of nonprotein nitrogen 30 

### (Atkinson et al., 1980). 31

32

### 8 DRAFT - DO NOT CITE OR QUOTE

3.2.1. Inhalation Exposure 1



### Little information was found in the available liter ature for distribution of inhaled 2 

### ammonia. Information on the distribution of endoge nously-produced ammonia suggests that any 3 

### ammonia absorbed through inhalation would be distri buted to all body compartments via the 4 

### blood, where it would be used in protein synthesis, as a buffer, reduced to normal concentrations 5 

### by urinary excretion, or converted by the liver to glutamine and urea (Takagaki et al., 1961). 6 

### Rats inhaling 300 ppm (212 mg/m 3 

### ) ammonia for 6 hours/day for 15 days exhibited 7 

### increased blood ammonia (200%) and brain (28%) glut amine levels at 5 days of exposure, but 8 

### not at 10 or 15 days (Manninen et al., 1988), demon strating transient distribution of ammonia to 9 

### the brain (metabolic adaptation). 10

11 

### 3.2.2. Oral Exposure 12 

### Human oral exposure data indicate that ammonia read ily enters the portal circulation and 13 

### is delivered to the liver, as has been shown to be the case for endogenously produced ammonia 14 

### (Pitts, 1971; Summerskill and Wolpert, 1970). Unio nized ammonia is freely diffusible, whereas 15 

### the ammonium ion is less so, and is relatively conf ined to the extracellular compartment 16 

### (Stabenau et al., 1958). 17 

### 3.2.3. Dermal Exposure 18 

### No quantitative data on distribution of ammonia fro m dermal exposure were located in 19



### the available literature. 20

21



### 3.3. METABOLISM 22 

### Endogenously, ammonia is produced by catabolism of amino acids by glutamate 23 

### dehydrogenase primarily in the liver and renal cort ex, but also in the brain and heart (Souba, 24 

### 1987). In skeletal muscle, ammonia may be produced by metabolism of adenosine 25 

### monophosphate (AMP) via adenylate deaminase. Infor mation on the metabolism of 26 

### exogenously-introduced ammonia was not found in the available literature. Ammonia and 27 

### ammonium ion are metabolized to glutamine mainly in the liver via glutamine synthetase in the 28 

### glutamine cycle (Figure 3-1), or incorporated into urea as part of the urea cycle as observed in 29 

### the hepatic mitochondria and cytosol (Figure 3-2) ( Souba, 1987). Ammonia can be rapidly 30 

### converted to glutamine in the brain as well (Takaga ki et al., 1961). Van de Poll (2008) reported 31



### that the liver removes an amount of ammonia from ci rculation equal to the amount added by the 32 

### intestines at metabolic steady state, indicating th at the gut does not contribute significantly to 33 

### systemic ammonia release. 34

### 9 DRAFT - DO NOT CITE OR QUOTE

1

2 3

4 

### Adapted from: Nelson and Cox (2008). 5

6 

### Figure 3-1. Glutamine cycle. 7

8 

### Glutamate

γ -Glutamyl phosphate glutamine synthetase

glutamine synthetase 

### Glutamine glutaminase (in liver mitochondria) NH 4 +

H 2 O ATP

ADP

NH 4 + P i

### 10 DRAFT - DO NOT CITE OR QUOTE

1

2 

### Adapted from: Nelson and Cox (2008). 3

4 

### Figure 3-2. The urea cycle showing the compartment alization of its steps 5 

### within liver cells. 6

7 

### Given its important metabolic role, ammonia exists in a homeostatically regulated 8 

### equilibrium in the body. In particular, free ammon ia has been shown to be homeostatically 9 

### regulated to remain at low concentrations, with 95– 98% of body burden existing in the blood (at 10 

### physiological pH) as NH 4 + 

### ion (da Fonseca-Wolhheim, 1995; Souba, 1987). Two studies in rats 11 

### (Manninen et al., 1988; Schaerdel et al., 1983) pro vided evidence that ammonia concentrations 12 

### in air below 25 ppm (18 mg/m 3 

### ) do not alter blood ammonia concentrations. Schae rdel et al. 13 

### (1983) exposed rats to ammonia for 24 hours at conc entrations ranging from 15–1,157 ppm (11- 14 

### 818 mg/m 3 

### ). Exposure to 15 ppm (11 mg/m 3 

### ) ammonia did not increase blood ammonia 15 

### concentrations after 24 hours; concentrations of ≥ 32 ppm caused an exposure-released increase 16 

### in blood ammonia, but concentrations at 12- and 24- hour sampling periods were lower than at 8 17 

### hours, suggesting compensation by increasing ammoni a metabolism through conversion to urea, 18 

### pyrimidine and polyamine synthesis, incorporation i nto amino acid substrates, and metabolism in 19 

### nervous system tissue. Rats inhaling 25 ppm (18 mg /m 3 

### ) ammonia for 6 hours/day for 15 days 20 Carbamoyl phosphate synthase I H 2 O

2 ATP

2 ADP

P i 3 H + CO 2 + 

### NH 4 +

P i



### Citrulline

Ornithine

Aspartate



### Argininosuccinate Arginine H 2 O Urea 

### Ornithine Ornithine transcarbamoylase



### Citrulline Carbamoyl phosphate

Argininosuccinate

synthase

Argininosuccinate lyase Arginase M ITOCHONDRIAL

M ATRIX



### Fumarate ATP

AMP + PP i

C YTOSOL

### 11 DRAFT - DO NOT CITE OR QUOTE

did not exhibit blood or brain ammonia or glutamine levels that were different from controls; 1 

### however, rats inhaling 300 ppm (212 mg/m 3 

### ) for the same duration exhibited statistically 2 

### significantly increased levels of blood ammonia (th reefold) and brain glutamine (approximately 3 

### 40%) at 5 days of exposure, but not at 10 or 15 day s (Manninen et al., 1988). The return of 4 

### blood and brain ammonia and glutamine levels to con trol levels with time is consistent with 5 

### metabolic adaptation, and these data suggest that a nimals have a large capacity to handle high 6



### concentrations of inhaled ammonia. 7 

### Various disease states can affect the rate of gluta mine uptake and catabolism and, 8 

### thereby, affect the blood and tissue levels of ammo nia. Abnormally elevated levels of ammonia 9 

### are indicative of end-stage renal failure (Davies e t al., 1997). Acute renal failure can result in 10 

### increased renal glutamine consumption and ammonia p roduction with a decreased capability of 11 

### eliminating urea in the urine (Souba, 1987). End-s tage liver failure due to fulminant hepatitis or 12 

### hepatic cirrhosis may result in decreased ureagenes is and increased levels of ammonia in blood 13 

### (hyperammonemia), leading to increased uptake into the brain and the onset of hepatic 14 

### encephalopathy. The increased metabolic alkalosis associated with hepatic encephalopathy may 15



### result in a shift in the NH 4 + 

### /NH 3 

### ratio in the direction of ammonia, which could pas s through the 16 

### blood-brain barrier (Katayama, 2004). In patients with liver cirrhosis and acute clinical hepatic 17 

### encephalopathy, the observed trapping of [ 13 

### N]-ammonia in the brain appeared to be related to a 18 

### fivefold increase, relative to healthy controls, of ammonia permeability across the blood-brain 19 

### barrier (Keiding et al., 2010, 2006). Furthermore, Sorensen et al. (2009) demonstrated greater 20



### unidirectional clearance of ammonia from the blood to brain cells than metabolic clearance of 21 

### ammonia from the blood in both healthy controls and in cirrhotic patients with and without 22 

### hepatic encephalopathy. 23

24



### 3.4. ELIMINATION 25 

### Absorbed ammonia, as well as endogenously produced ammonia, is excreted by the 26 

### kidneys as urea (Summerskill and Wolpert, 1970; Gay et al., 1969; Muntwyler et al., 1956; 27 

### Davies and Yudkin, 1952; Van Slyke et al., 1943) an d is a component of sweat (Guyton, 1981; 28 

### Wands, 1981). Lee and colleagues observed that acidosis-stimulated renal excretion o f ammonia 29 

### is mediated by intercalated cell-specific Rh B glyc oprotein expression in mice (Bishop et al., 30 

### 2010; Lee et al., 2010, 2009). In rat kidney, ammonium ion is secreted into the lu men of the 31 

### outer medullary collecting duct via H + 

### secretion and parallels ammonia diffusion (Flessne r et al., 32 

### 1992). The inner medullary collecting duct exhibit s a Na + 

### - and K + 

### -independent NH 4 + 

### /H + 33 

### exchange activity that may be mediated by an Rh C g lycoprotein (Handlogten et al., 2005), 34 

### which is also expressed in human kidneys (Han et al ., 2006). 35 

### Additionally, ammonia is known to be excreted throu gh expired air and is present in the 36 

### expired air of all humans (Manolis, 1983). Two inv estigators specifically measured ammonia in 37 

### breath exhaled from the nose (Smith et al., 2008; L arson et al., 1977). Smith et al. (2008) 38

### 12 DRAFT - DO NOT CITE OR QUOTE

reported median ammonia concentrations in exhaled b reath from the nose of three healthy 1 

### volunteers (with samples collected daily over a 4-w eek period) of 0.059–0.078 mg/m 3 

### ; these 2 

### concentrations were similar to or slightly higher t han the mean laboratory air level of ammonia 3 

### of 0.056 mg/m 3 

### reported in this study. Larson et al. (1977) repo rted that the median 4 

### concentration of ammonia collected from air samples exhaled from the nose ranged from 0.013 5 

### to 0.046 mg/m 3 

### . One sample collected from the trachea (via a tub e inserted through the nose of 6 

### one subject) was 0.029 mg/mg 3 

### —a concentration within the range of concentrations in breath 7 

### exhaled through the nose (Larson et al., 1977). 8 

### Higher and more variable ammonia concentrations ar e reported in breath exhaled from 9 

### the mouth or oral cavity than from air exhaled from the nose. In studies that reported ammonia 10 

### in breath samples from the mouth or oral cavity, th e majority of ammonia concentrations ranged 11 

### from 0.085 to 2.1 mg/m 3 

### (Smith et al., 2008; Spanel et al., 2007a; Spanel et al., 2007b; Turner et 12 

### al., 2006; Diskin et al., 2003; Smith et al., 1999; Norwood et al., 1992; Larson et al., 1977). 13 

### These higher concentrations are largely attributed to the production of ammonia by bacterial 14 

### degradation of food protein in the oral cavity or G I tract (Turner et al., 2006; Smith et al., 1999; 15 

### Vollmuth and Schlesinger, 1984). This source of am monia in breath was demonstrated by Smith 16 

### et al. (1999), who observed elevated ammonia concen trations in the expired air of 6 healthy 17 

### volunteers following the ingestion of a protein-ric h meal. 18



### Other factors that can affect ammonia levels in bre ath exhaled from the mouth or oral 19 

### cavity include diet, oral hygiene, age, living cond itions, and disease state. Norwood et al. (1992) 20 

### reported decreases in baseline ammonia levels (0.08 5–0.905 mg/m 3 

### ) in exhaled breath following 21 

### tooth brushing (<50% depletion), a distilled water oral rinse (<50% depletion), and an acid oral 22 

### rinse (80–90% depletion). These findings are consi stent with ammonia generation in the oral 23 

### cavity by bacterial and/or enzymatic activity. Sev eral investigators have reported that ammonia 24 

### in breath from the mouth and oral cavity increases with age (Spanel et al., 2007a; Spanel et al., 25 

### 2007b; Turner et al., 2006), with ammonia concentra tions increasing, on average, by about 0.1 26 

### mg/m 3 

### for each 10 years of life (Spanel et al., 2007b). Turner et al. (2006) reported that the age 27



### of the individual accounts for about 25% of the var iation in observed mean breath ammonia 28 

### levels and the remaining 75% is due to factors othe r than age. Certain disease states can also 29



### influence ammonia levels in exhaled breath. Ammoni a is greatly elevated in the breath of 30 

### patients in renal failure (Spanel et al., 2007b; Da vies, 1997). Increases in breath ammonia are 31 

### associated with decreasing kidney function with inc reasing age (Diskin et al., 2003; Epstein, 32 

### 1996). These studies are further described in Appe ndix C.1 in Table C-1. 33 

### Because ammonia measured in samples of breath exhal ed from the mouth or oral cavity 34 

### can be generated in the oral cavity and thus may be substantially influenced by diet and other 35 

### factors, ammonia levels measured in mouth or oral c avity breath samples do not likely reflect 36 

### systemic (blood) levels of ammonia. Ammonia concen trations in breath exhaled from the nose 37 

### appear to better represent systemic or background l evels (Smith et al., 2008). 38

### 13 DRAFT - DO NOT CITE OR QUOTE

Ammonia has also been detected in the expired air o f animals. Whittaker et al. (2009) 1 

### observed a significant association between ambient ammonia concentrations and increases in 2 

### exhaled ammonia in stabled horses. Analysis of end ogenous ammonia levels in the expired air 3 

### of rats showed concentrations ranging from 0.01 to 0.353 ppm (0.007–0.250 mg/m 3 

### ) 4 

### (mean = 0.08 ppm or 0.06 mg/m 3 

### ) in nose-breathing animals (Barrow and Steinhagen, 1980). 5 

### Larson et al. (1980) reported ammonia concentration s measured in the larynx of dogs exposed to 6 

### sulfuric acid ranging between 0.03 and 0.225 ppm (0 .02 and 0.16 mg/m 3 

### ) following mouth 7 

### breathing, and between 0.05 and 0.220 ppm (0.04 and 0.16 mg/m 3 

### ) following nose breathing. 8

9



### 3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS 10 

### No physiologically based pharmacokinetic (PBPK) mod els have been developed for 11 

### ammonia. An expanded one-compartment toxicokinetic model in rats was developed by Diack 12 

### and Bois (2005), which used physiological values to represent first-order uptake and elimination 13 

### of inhaled ammonia (and other chemicals). The mode l is not useful for dose-response 14 

### assessment of ammonia because: (1) it cannot speci fy time-dependent amounts or concentrations 15 

### of ammonia in specific target tissues, (2) it has n ot been verified against experimental data for 16 

### ammonia, glutamate, or urea levels in tissues, and (3) it cannot extrapolate internal doses of 17



### ammonia between animals and humans. 18

19

### 14 DRAFT - DO NOT CITE OR QUOTE 4. HAZARD IDENTIFICATION 1

2

3 

### As noted in Section 2, ammonium salts (e.g., ammoni um acetate, chloride, and sulfate) 4 

### readily dissolve in water through disassociation in to the ammonium ion (NH 4 + 

### ) and the anion. 5 

### At physiological pH (7.4), the equilibrium favors t he formation of NH 4 + 

### . Because of this 6 

### equilibrium at physiological pH, the literature on the toxicity of ammonium salts was reviewed 7



### to determine whether this literature could inform t he toxicity of ammonia. In rats following 8 

### exposure to ammonium chloride in the diet for subch ronic and chronic exposure durations at 9 

### doses ranging from approximately 500 to 1,800 mg/kg -day, the primary effect of this salt was 10 

### related to the acid-base balance in the body (Lina and Kuijpers, 2004; Barzel et al., 1969). 11



### Ammonium chloride treatment induced a dose-related hyperchloremic metabolic acidosis in rats 12 

### as evidenced by decreases in blood pH, base excess, and bicarbonate concentration, and 13 

### increased plasma chloride levels. Lina and Kuijper s (2004) also observed a significant dose14 

### related depression in body weight gain at exposure to ammonium chloride doses ≥ 1,590 mg/kg15 

### day for 13 weeks. The administration of ammonium c hloride was also associated with zona 16 

### glomerulosa hypertrophy of the adrenal gland in bot h the 13-week and 18- and 30-month studies 17 

### by Lina and Kuijpers (2004). 18 

### In contrast, metabolic acidosis was not induced in rats exposed to ammonium sulfate in 19 

### the diet at doses up to 1,527 mg/kg-day for 52 week s or histopathologic changes in the adrenal 20 

### gland at doses up to 1,371 mg/kg-day for 104 weeks (Ota et al., 2006). The only dose-related 21 

### effects associated with the 52-week exposure to amm onium sulfate were increased liver and 22 

### kidney weights (~7 and 10%, respectively) at a dose of 1,490 mg/kg-day (female rat) or 1,527 23 

### mg/kg-day (male rat) (Ota et al., 2006). In the 10 4-week study, the incidence of chronic 24 

### nephropathy was statistically significantly increas ed in low-dose (564 mg/kg-day), but not in 25 

### high-dose (1,288 mg/kg-day) male rats (Ota et al., 2006). 26 

### Appendix B, Table B-1, presents a summary of repeat -dose oral toxicity studies for 27



### selected ammonium salts. Studies of ammonium sulfa mate, a broad spectrum herbicide, were 28 

### not included in this summary table because the pest icidal properties of this ammonium salt were 29 

### not considered relevant to ammonia toxicity. No st udies of the toxicity of ammonium salts by 30 

### the inhalation pathway are available. 31 

### Because the toxicity information for the ammonium s alts that have been adequately 32 

### studied (i.e., the chloride and sulfate) indicates that their toxicity profiles differ, it appears tha t 33 

### the anion can influence the toxicity of the ammoniu m salt. Therefore, it is uncertain whether 34 

### toxicity data for ammonium salts can be used to inf orm the toxicity of ammonia. Accordingly, 35 

### the toxicity of ammonium salts as the basis for cha racterizing the toxicity of ammonia was not 36



### further evaluated in this assessment. 37

38

### 15 DRAFT - DO NOT CITE OR QUOTE

4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL 1



### CONTROLS 2 

### 4.1.1. Case Reports – Oral and Inhalation Exposure s 3 

### Ammonia exposure has occurred frequently in occupat ional settings (both industrial and 4 

### agricultural) when equipment failure or operator er ror resulted in the sudden release of 5 

### pressurized ammonia. Transportation accidents and catastrophic releases have also resulted in 6 

### exposure to high concentrations of ammonia. Oral e xposure to ammonia has resulted from 7 

### intentional or accidental ingestion of household pr oducts containing ammonia. Numerous case 8 

### reports of injury in adults and children due to exp osure to ammonia via inhalation of vapors, 9 

### dermal contact, or ingestion of household cleaning solutions or ammonia inhalant capsules exist. 10 

### These case reports indicate that the clinical sign s of acute oral exposure to ammonia 11 

### were headache, stomachache, nausea, dizziness, diar rhea, drooling, erythematous and edematous 12 

### lips, reddened and blistered tongues, dysphagia, vo miting, oropharyngeal burns, laryngeal and 13 

### epiglottal edema, erythmatous esophagus with severe corrosive injury, and hemorrhagic 14 

### esophago-gastro-duodeno-enteritis. Acute inhalatio n or dermal exposure to ammonia resulted in 15 

### first-, second-, and third-degree burns to body sur faces, mild to severe erythema and edema 16 

### throughout the mouth, nasal passages, pharynx, lary nx, trachea and esophagus, mild to severe 17 

### respiratory distress with diffuse or scattered rale s and rhonchi, hypoxemia, wheezing, and upper 18 

### airway obstruction, eye irritation, blepharospasm, lacrimation, ocular erythema, edema, and 19 

### death. In addition, delayed effects and complicati ons involving the eyes and respiratory system 20 

### were often encountered even after initial improveme nt in acute conditions. Delayed effects 21 

### included complete or partial loss of vision, persis tent and/or progressive shortness of breath 22 

### (especially on exertion), persistent, productive co ugh, and progressive obstructive and restrictive 23 

### pulmonary disease with development of cylindrical a nd/or saccular bronchiectasis with or 24 

### without obliterative fibrous adhesions in the pleur al space, and death. The details of these case 25 

### reports are given in Appendix C.2. 26

27 

### 4.1.2. Controlled Human Inhalation Exposure Studie s 28 

### Several controlled exposure studies were conducted in volunteers to evaluate irritation 29 

### effects and changes in pulmonary function following acute inhalation exposure to ammonia; 30 

### some of these studies describe the occurrence of ey e, nose, and throat irritation. These studies 31 

### are presented in more detail in Appendix C.3. Some of the studies included in Appendix C.3 did 32 

### not provide information on the human subjects resea rch ethics procedures undertaken in the 33 

### study (Altmann et al., 2006 (abstract only); Dougla s and Coe, 1987; Kalandarov et al., 1984; 34 

### Ferguson et al., 1977; Verberk, 1977; Silverman et al., 1949), but there is no evidence that the 35 

### conduct of the research was fundamentally unethical or significantly deficient relative to the 36 

### ethical standards prevailing at the time the resear ch was conducted. Other studies reported that 37 

### informed consent by volunteers and/or study approva l by local boards/officials regarding ethical 38

### 16 DRAFT - DO NOT CITE OR QUOTE

conduct was obtained (Petrova et al., 2008; Smeets et al., 2007; Ihrig et al., 2006; Sigurdarson et 1 

### al., 2004; Sundblad et al., 2004; Cole et al., 1977 ; MacEwan et al., 1970). 2 

### Altmann et al. (2006) showed a dose-dependent incre ase in the intensity of odor 3 

### annoyance and irritation in healthy male and female volunteers during inhalation exposure to 4 

### ammonia; strong olfactory and moderate to strong ir ritation sensations occurred at concentrations 5 

### >15 ppm (11 mg/m 3 

### ), with odor detection thresholds at <20 ppm (14 mg /m 3 

### ). In another study, 6 

### 12 healthy volunteers exposed to 5 and 25 ppm (4 an d 18 mg/m 3 

### ) ammonia on three different 7 

### occasions for 1.5 hours in an exposure chamber whil e exercising on a stationary bike reported 8 

### discomfort in the eyes and odor detection at 5 ppm (4 mg/m 3 

### ) (Sundblad et al., 2004). Eye 9



### irritation was also shown to increase in a concentr ation-dependent manner in 15 volunteers 10 

### exposed to ammonia for 2 hours in an exposure chamb er at concentrations of 50, 80, 110, and 11 

### 140 ppm (35, 57, 78, and 99 mg/m 3 

### ); ammonia concentrations of 140 ppm (99 mg/m 3 

### ) caused 12 

### severe and intolerable irritation (Verberk, 1977). The lachrymatory threshold was determined to 13 

### be 55 ppm (39 mg/m 3 

### ) in volunteers exposed to ammonia gas inside tight -fitting goggles for an 14 

### acute duration of up to 15 seconds (Douglas and Coe , 1987). In contrast, exposures to up to 90 15 

### ppm (64 mg/m 3 

### ) ammonia gas did not produce severe lacrimation in seven volunteers after 10 16 

### minutes in an exposure chamber, although increased eye erythema was reported (MacEwen et 17 

### al., 1970). Exposure to 500 ppm (354 mg/m 3 

### ) of ammonia gas for 30 minutes through a masked 18 

### nose and throat inhalation apparatus resulted in 2/ 7 volunteers reporting lacrimation, and 2/7 19 

### reporting nose and throat irritation that lasted up to 24 hours after exposure (Silverman et al., 20 

### 1949). 21 

### Petrova et al. (2008) investigated irritation thres hold differences between 25 healthy 22 

### volunteers and 15 mild-to-moderate persistent asthm atic volunteers exposed to ammonia via the 23 

### eyes and nose at concentrations ranging from 2 to 5 00 ppm (1–354 mg/m 3 

### ) for durations lasting 24 

### up to 2.5 hours. Irritation threshold, odor intens ity, and annoyance were not significantly 25 

### different between the two groups. The nasal and ey e irritation thresholds were reported to be 26 

### 129 ppm (91 mg/m 3 

### ) and 175 ppm (124 mg/m 3 

### ), respectively. Smeets et al. (2007) investigated 27 

### odor and irritation thresholds for ammonia vapor in 24 healthy female volunteers at 28 

### concentrations ranging from 0.03 to 615 ppm (0.02 t o 435 mg/m 3 

### ). This study observed a mean 29 

### odor detection threshold of 2.6 ppm (2 mg/m 3 

### ) and a mean irritation threshold of 31.7 or 60.9 30 

### ppm (22 or 43 mg/m 3 

### ), depending on the olfactometry methodology follow ed (static versus 31 

### dynamic, respectively). Irritation thresholds may be higher in people who have had prior 32 

### experience with ammonia exposure (Ihrig et al., 200 6). Thirty male volunteers who had not 33 

### experienced the smell of ammonia and 10 male volunt eers who had regular workplace exposure 34 

### to ammonia were exposed to ammonia vapors at concen trations of 0, 10, 20, and 50 ppm (0, 7, 35 

### 14, and 35 mg/m 3 

### ) on 5 consecutive days (4 hours/day) in an exposur e chamber; volunteers in 36 

### the group familiar to the smell of ammonia reported fewer symptoms than the nonhabituated 37 

### group, but at a concentration of 20 ppm (14 mg/m 3 

### ), there were no differences in perceived 38

### 17 DRAFT - DO NOT CITE OR QUOTE

symptoms between the groups. However, the perceive d intensity of symptoms was 1 

### concentration-dependent in both groups, but was onl y significant in the group of volunteers not 2 

### familiar with ammonia exposure (Ihrig et al., 2006) . Ferguson et al. (1977) reported habituation 3 

### to eye, nose, and throat irritation in six male and female volunteers after 2–3 weeks of exposure 4 

### to ammonia concentrations of 25, 50, and 100 ppm (1 8, 35, and 71 mg/m 3 

### ) during a 6-week 5 

### study (6 hours/day, 1 time/week). Continuous expos ure to even the highest concentration tested 6 

### became easily tolerated with no general health effe cts occurring after acclimation occurred. 7 

### Several studies evaluated pulmonary functions follo wing acute inhalation exposure to 8 

### ammonia. Volunteers exposed to ammonia (lung only) through a mouthpiece for 10 inhaled 9 

### breaths of gas experienced bronchioconstriction at a concentration of 85 ppm (60 mg/m 3 

### ) 10 

### (Douglas and Coe, 1987); however, there were no bro nchial symptoms reported in seven 11 

### volunteers exposed to ammonia at concentrations of 30, 50, or 90 ppm (21, 35, and 64 mg/m 3 

### ) 12 

### for 10 minutes in an exposure chamber (MacEwen et a l., 1970). Similarly, 12 healthy volunteers 13 

### exposed to ammonia on three separate occasions to 5 and 25 ppm (4 and 18 mg/m 3 

### ) for 1.5 hours 14 

### in an exposure chamber while exercising on a statio nary bike did not have changes in bronchial 15 

### responsiveness, upper airway inflammation, exhaled nitric oxide levels, or lung function as 16 

### measured by vital capacity and FEV 1 

### (Sundblad et al., 2004). In another study, 18 hea lthy 17 

### servicemen volunteers were placed in an exposure ch amber for 3 consecutive half-day sessions. 18 

### Exposure to ammonia at concentrations of 50–344 mg/ m 3 

### (70–486 ppm) occurred on the 2 nd 19 

### session, with sessions 1 and 3 acting as controls ( Cole et al., 1977). The no-effect concentration 20 

### was determined to be 71 mg/m 3 

### (100 ppm). Exercise tidal volume was increased at 106 mg/m 3 21 

### (150 ppm), but then decreased at higher concentrati ons in a concentration-dependent manner 22 

### (Cole et al., 1977). Decreased FEV 1 

### and forced vital capacity (FVC) were reported in e ight 23 

### healthy male volunteers exposed to a mean airborne ammonia concentration of 20.7 ppm (15 24 

### mg/m 3 

### ) in swine confinement buildings for 4 hours at one -week intervals; however, swine 25 

### confinement buildings also include confounding expo sures to dust, bacteria, endotoxin, and 26 

### molds, thereby making measurement of effects due to ammonia uncertain in this study (Cormier 27 

### et al., 2000). 28 

### Differences in pulmonary function between healthy a nd asthmatic volunteers exposed to 29



### ammonia were evaluated in several studies. There w ere no changes in lung function as measured 30 

### by FEV 1 

### in 25 healthy volunteers and 15 mild/moderate persi stent asthmatic volunteers after 31 

### ocular and nasal exposure to 2–500 ppm (1–354 mg/m 3 

### ) ammonia at durations lasting up to 32 

### 2.5 hours (Petrova et al., 2008). In another study , six healthy volunteers and eight mildly 33 

### asthmatic volunteers were exposed to 16–25 ppm (11– 18 mg/m 3 

### ) ammonia, ammonia and dust, 34 

### and dust alone for 30-minute sessions, with 1 week between sessions (Sigurdarson et al., 2004). 35 

### There were no significant changes in pulmonary func tion as measured by FEV 1 

### in the healthy 36 

### volunteers for any exposure. A decrease in FEV 1 

### was reported in asthmatics exposed to dust and 37 

### ammonia, but not ammonia alone; similarly, increase d bronchial hyperreactivity was reported in 38

### 18 DRAFT - DO NOT CITE OR QUOTE

asthmatics after exposure to dust and ammonia, but not to ammonia alone. Exposure to dust 1 

### alone caused similar effects, suggesting that dust was responsible for decreased pulmonary 2 

### function (Sigurdarson et al., 2004). 3 

### Kalandarov et al. (1984) investigated the effect of ammonia exposure on the function of 4 

### the sympathico-adrenal system and the adrenal corte x in male volunteers and found that 5 

### exposure to 2 mg/m 3 

### (3.0 ppm) ammonia in combination with increased te mperature and 6 

### humidity did not result in any significant changes to the function of the sympathico-adrenal 7 

### system, although the adrenal cortex function was af fected exhibiting increased 11- 8 

### oxycorticosteroids in plasma. Ammonia concentratio ns of 5 mg/m 3 

### (7.2 ppm), in combination 9 

### with increased temperature and humidity, resulted i n increased levels of adrenaline, 17- 10 

### oxycorticosteroids, and free 11-oxycorticosteroids fraction in plasma. These results suggest that 11 

### both sympathico-adrenal system and adrenal cortex f unction is altered at an ammonia 12 

### concentration of 5 mg/m 3 

### (7.2 ppm) (Kalandarov et al., 1984). 13 

### In summary, volunteer studies demonstrate that eye irritation can occur following acute 14 

### exposure to ammonia at concentrations as low as 5 p pm (4 mg/m 3 

### ). Irritation thresholds may be 15 

### higher in people who have had prior experience with ammonia exposure and habituation to eye, 16 

### nose, and throat irritation occurs over time. Pulm onary function was not affected in workers 17 

### acutely exposed to ammonia concentrations as high a s 71 mg/m 3 

### (100 ppm). Studies comparing 18 

### the pulmonary function of asthmatics and healthy vo lunteers exposed to ammonia do not suggest 19 

### that asthmatics are more sensitive to the pulmonary effects of ammonia. 20

21 

### 4.1.3. Cross Sectional Studies in Farmers Exposed to Inhaled Ammonia 22 

### Several studies have evaluated respiratory symptoms and changes in pulmonary function 23 

### in livestock farmers and stable workers exposed to ammonia (see Appendix C.4 for detailed 24 

### descriptions). In addition to ammonia, these studi es also documented exposures to airborne dust, 25 

### bacteria, fungal spores, endotoxin, and mold. The release of other volatiles on livestock farms is 26 

### likely, but measurements for other volatile chemica ls were not conducted. Although studies of 27 

### farm workers summarized here focused on exposure to ammonia, these and other studies have 28 

### also demonstrated respiratory effects associated wi th exposure to other constituents in farm 29 

### worker air (e.g., respirable dust, endotoxin). 30 

### Swine and dairy farmers had a higher prevalence of respiratory symptoms including 31 

### cough, phlegm, wheezing, chest tightness, and eye, nasal and throat irritation compared to 32 

### controls (Melbostad and Eduard, 2001; Preller et al ., 1995; Choudat et al., 1994; Zejda et al., 33 

### 1994; Crook et al., 1991; Heederik et al., 1990). Impaired respiratory function in farmers was 34 

### associated with ammonia exposure in several studies (e.g., decreased FEV 1 

### , FVC) (Cormier et 35 

### al., 2000; Donham et al., 2000, 1995; Vogelzang et al., 1998; Reynolds et al., 1996; Preller et al., 36 

### 1995; Crook et al., 1991; Heederik et al., 1990). Bronchial hyperreactivity to methacholine or 37 

### histamine challenge was increased in farmers expose d to ammonia compared to control workers 38

### 19 DRAFT - DO NOT CITE OR QUOTE

(Vogelzang et al., 2000, 1997; Choudat et al., 1994 ). Stable workers showed signs of bronchial 1 

### obstruction with increased peak expiratory flow (PE F) variability as well as increased pulmonary 2 

### inflammation related to allergies (Elfman et al., 2 009). Other findings that suggest an allergic or 3 

### inflammatory response in livestock farmers exposed to ammonia include the presence of 4 

### immunoglobin E (IgE) and immunoglobin G (IgG) antib odies to pig squames and urine in blood 5 

### (Crook et al., 1991), increased neutrophils in the nasal wash (Cormier et al., 2000) and increased 6 

### white blood cell count (Cormier et al., 2000). In summary, several studies have demonstrated an 7 

### association between ammonia exposure in livestock f armers and respiratory symptoms and 8 

### impaired respiratory function; however, farmers are additionally exposed to several constituents 9 

### that likely contribute to these effects, including respirable dust, endotoxin, bacteria, fungi, and 10



### mold. 11

12 

### 4.1.4. Occupational Studies in Industrial Worker P opulations 13 

### Holness et al. (1989) conducted a cross-sectional s tudy of workers in a soda ash (sodium 14 

### carbonate) plant 3 

### who had chronic low-level exposure to ammonia. Th e cohort consisted of 15



### 58 workers and 31 controls from stores and office a reas of the plant. All workers were males 16 

### (average age 40.5 years) and the average exposure d uration for the exposed workers at the plant 17 

### was 12.2 years. The mean time-weighted average (TW A) ammonia exposure of the exposed 18 

### group based on personal sampling over one work shif t (mean sample collection time, 8.4 hours) 19 

### was 9.2 ppm (6.5 mg/m 3 

### ), compared to 0.3 ppm (0.2 mg/m 3 

### ) for the control group. The average 20 

### concentrations of ammonia to which workers were exp osed were determined using the procedure 21 

### recommended by the National Institute for Occupatio nal Safety and Health (NIOSH) which 22 

### involves the collection of air samples on sulfuric acid-treated silica gel (ATSG) adsorption tubes 23 

### (NIOSH, 1979). 24 

### No statistically significant differences were obser ved in age, height, years worked, 25 

### percentage of smokers, or pack-years smoked for exp osed versus control workers. Exposed 26 

### workers weighed approximately 8% ( p < 0.05) more than control workers. Information 27 

### regarding past occupational exposures, working cond itions, and medical and smoking history, as 28 

### well as respiratory symptoms and eye and skin compl aints was obtained by means of a 29 

### questionnaire that was based on an American Thoraci c Society questionnaire (Ferris, 1978). 30 

### Each participant’s sense of smell was evaluated at the beginning and end of the work week using 31 

### several concentrations of pyridine (0.4, 0.66, or 1 0 ppm). Lung function tests were conducted at 32 

### the beginning and end of the work shift on the firs t and last days of their work week (four tests 33 

### administered). Differences in reported symptoms an d lung function between groups were 34 

### evaluated using the actual exposure values with age , height, and pack-years smoked as covariates 35

3 At this plant, ammonia, carbon dioxide, and water w ere the reactants used to form ammonium bicarbonate , which in turn was reacted with salt to produce sodium bic arbonate and subsequently processed to form sodium carbonate. Ammonia and carbon dioxide were recoved in the proc ess and reused.

### 20 DRAFT - DO NOT CITE OR QUOTE

in linear regression analysis. Exposed workers wer e grouped into three exposure categories 1 

### (high = >12.5 ppm [>8.8 mg/m 3 

### ], medium = 6.25–12.5 ppm [4.4–8.8 mg/m 3 

### ], and low = 2 

### <6.25 ppm [<4.4 mg/m 3 

### ]) for analysis of symptom reporting and pulmonary function data. 3 

### Endpoints evaluated in the study included sense of smell, prevalence of respiratory 4 

### symptoms (cough, bronchitis, wheeze, dyspnea, and o thers), eye and throat irritation, skin 5 

### problems and lung function parameters (FVC, FEV 1 

### , FEV 1 

### /FVC, forced expiratory flow [FEF 50 

### ], 6



### and FEF 75 

### ). No statistical differences in the prevalence of respiratory symptoms or eye irritation 7 

### were evident between the exposed and control groups (Table 4-1). There was a statistically 8 

### significant increase (p < 0.05) in the prevalence o f skin problems in workers in the lowest 9 

### exposure category (<4.4 mg/m 3 

### ) compared to controls; however, the prevalence was not 10 

### increased among workers in the two higher exposure groups. Workers also reported that 11 

### exposure at the plant had aggravated specific sympt oms including coughing, wheezing, nasal 12 

### complaints, eye irritation, throat discomfort, and skin problems. Odor detection threshold and 13 

### baseline lung functions were similar in the exposed and control groups. No changes in lung 14 

### function were demonstrated over either work shift ( days 1 or 2) or over the work week in the 15 

### exposed group compared with controls. No relations hip was demonstrated between chronic 16 

### ammonia exposure and baseline lung function changes either in terms of the level or duration of 17 

### exposure. Study investigators noted that this find ing was limited by the lack of adequate 18 

### exposure data collected over time, precluding devel opment of a meaningful index accounting for 19 

### both level and length of exposure. Based on the la ck of exposure-related differences in 20 

### subjective symptomatology, sense of smell, and meas ures of lung function, EPA identified 8.8 21 

### mg/m 3 

### (12.5 ppm) as the no-observed-adverse-effect level (NOAEL). A lowest-observed22 

### adverse-effect level (LOAEL) was not identified for this study. 23

24

### 21 DRAFT - DO NOT CITE OR QUOTE

Table 4-1. Symptoms and lung function results of w orkers exposed to different levels of TWA ammonia concentrations

Parameter Ammonia concentration

Control 0.2 mg/m 3 (0.3 ppm) Exposed

<4.4 mg/m 3 (<6.25 ppm) Exposed

4.4–8.8 mg/m 3 (6.25–12.5 ppm) Exposed

>8.8 mg/m 3 (>12.5 ppm) Symptom Cough 3/31(10) a 6/34 (18) 1/12 (8) 2/12 (17) Sputum 5/31 (16) 9/34 (26) 3/12 (25) 1/12 (8) Wheeze 3/31 (10) 5/34 (15) 1/12 (8) 0/12 (0) Chest tightness 2/31 (6) 2/34 (6) 0/12 (0) 0/12 (0) Shortness of breath 4/31 (13) 3/34 (9) 1/12 (8) 0/12 (0) Nasal complaints 6/31 (19) 4/34 (12) 2/12 (17) 0/12 (0) Eye irritation 6/31 (19) 2/34 (6) 2/12 (17) 1/12 (8 ) Throat irritation 1/31 (3) 2/34 (6) 1/12 (8) 1/12 ( 8) Skin problems 2/31 (6) 10/34 b (29) 1/12 (8) 1/12 (8) Lung function (% predicted)

FVC 98.6 96.7 96.9 96.8

FEV 1 95.1 93.7 93.9 95.3

FEF 50 108.4 106.9 106.2 111.2

FEF 75 65.2 71.0 67.8 78.8

FVC = forced vital capacity; FEV 1 =forced expiratory volume in 1 second; FEF 50 =forced expiratory flow rates at 50%; FEF 75 =forced expiratory flow rates at 75%

a Number affected/number examined. The percentage of workers reporting symptoms is indicated in parenthe ses. b Significantly different from controls, p < 0.05, by Fisher’s exact test performed for this review.

Source: Holness et al. (1989).

1 

### Ballal et al. (1998) conducted a cross-sectional st udy of male workers at two urea 2



### fertilizer factories in Saudi Arabia 4 

### . The cohort consisted of 161 exposed subjects (84 from 3 

### factory A and 77 from factory B) and 355 unexposed controls. Workers in factory A were 4 

### exposed to air ammonia levels of 2–130 mg/m 3 

### (2.8–184.4 ppm), and workers in factory B were 5 

### exposed to 0.02–7 mg/m 3 

### (0.03–9.9 ppm). Mean duration of employment was 51 .8 months for 6 

### exposed workers and 73.1 months for controls. Expo sure levels were estimated by analyzing a 7 

### total of 97 air samples collected over 8-hour shift s close to the employee’s work site. The 8 

### prevalence of respiratory symptoms and diseases was determined by administration of a 9 

### questionnaire. The authors stated that there were no other chemical pollutants in the workplace 10 

### that might have affected the respiratory system. S moking habits were similar for exposed 11 

### workers and controls. Stratifying the workers by a mmonia exposure levels (above or below the 12 

### American Conference of Governmental Industrial Hygi enists [ACGIH] threshold limit value 13

4 The process of fertilizer production involved synth esis of ammonia from natural gas, followed by react ion of the ammonia and carbon dioxide to form ammonium carbami de, which was then converted to urea.

### 22 DRAFT - DO NOT CITE OR QUOTE

[TLV] of 18 mg/m 3 

### [25 ppm]) showed that those exposed to ammonia conc entrations higher than 1 

### the TLV had significantly higher relative risks for cough, phlegm, wheezing, dyspnea, and 2 

### asthma than workers exposed to levels below the TLV (Table 4-2). The relative risk for 3 

### wheezing was also elevated among those exposed to a mmonia levels at or below the TLV. 4 

### Distribution of symptoms by cumulative ammonia conc entration (CAC, mg/m 3 

### -years) also 5 

### showed significantly higher relative risk for all t he above symptoms among those with higher 6 

### CAC (Table 4-2). Results of the logistic regressio n analysis showed that ammonia concentration 7 

### was significantly related to cough, phlegm, wheezin g with and without shortness of breath, and 8 

### asthma (Table 4-3). 9

10 

### Table 4-2. The prevalence of respiratory symptoms and disease in urea

fertilizer workers exposed to ammonia

Respiratory symptom/disease Relative risk (95% CI)

Exposure category Cumulative concentration (mg/m 3 of air-yrs)

≤ ACGIH TLV (18 mg/m 3 ) (n = 138) >ACGIH TLV

(18 mg/m 3 ) (n = 17) ≤ 50

(n = 130) >50

(n = 30) Cough 0.86 (0.48–1.52) 3.48 (1.84–6.57) 0.72 (0.38– 1.35) 2.82 (1.58–5.03) Wheezing 2.26 (1.32–3.88) 5.01 (2.38–10.57) 1.86 (1 .04–3.32) 5.24 (2.85–9.52) Phlegm 0.79 (0.43–1.47) 3.75 (1.97–7.11) 0.63 (0.31 –1.26) 3.03 (1.69–5.45) Dyspnea 1.13 (0.62–2.04) 4.57 (2.37–8.81) 1.19 (0.6 6–2.17) 2.59 (1.25–5.36) Chronic bronchitis 1.43 (0.49–4.19) 2.32 (0.31–17.2 8) 0.61 (0.13–2.77) 5.32 (1.72–16.08) Bronchial asthma 1.15 (0.62–2.15) 4.32 (2.08–8.98) 1.22 (0.66–2.28) 2.44 (1.10–5.43)

Chronic bronchitis and bronchial asthma 2.57 (0.53–12.59) 6.96 (0.76–63.47) 1.82 (0.31–10.7 7) 8.38 (1.37–45.4)

Source: Ballal et al. (1998).

11 

### Table 4-3. Logistic regression analysis of the rel ationship between ammonia

concentration and respiratory symptoms or disease i n exposed urea fertilizer workers

Respiratory symptom/disease OR (95% CI) Cough 1.32 (1.08–1.62) a Phlegm 1.36 (1.10–1.67) a Shortness of breath with wheezing 1.26 (1.04–1.54) a Wheezing alone 1.55 (1.17–2.06) a Dyspnea on effort 0.83 (0.68–1.02) Diagnosis of asthma 1.33 (1.07–1.65) a

a p ≤ 0.05.

Source: Ballal et al. (1998).

### 23 DRAFT - DO NOT CITE OR QUOTE

Results from limited spirometry testing of workers from factory A were reported in a 1 

### followup study (Ali, 2001). The pulmonary function indices measured in 73 ammonia workers 2



### and 343 control workers included FEV 1 

### and FVC. Prediction equations for these indices w ere 3 

### developed for several nationalities (Saudis, Arabs, Indians, and other Asians) and corrected 4 

### values were expressed as the percentage of the pred icted value for age and height. The FVC% 5 

### predicted was higher in exposed workers than in con trols (4.6% increase, p ≤ 0.002); however, 6 

### workers with cumulative exposure ≥ 50 mg/m 3 

### -years had significantly lower FEV 1 

### % predicted 7 

### (7.4% decrease, p < 0.006) and FVC% predicted (5.4% decrease, p ≤ 0.030) than workers with 8 

### cumulative exposure ≤ 50 mg/m 3 

### -years. A comparison between symptomatic and asymp tomatic 9 

### exposed workers showed that FEV 1 

### % predicted and FEV 1 

### /FVC% were significantly lower 10 

### among symptomatic workers (9.2% decrease in FEV 1 

### % predicted, p < 0.001 and 4.6% decrease 11



### in FEV 1 

### /FVC%, p < 0.02). Although Ballal et al. (1998) and Ali (2 001) suggest that exposure to 12 

### ammonia concentrations above 18 mg/m 3 

### (50 mg/m 3 

### -years) is associated with respiratory 13 

### symptoms and altered pulmonary function, NOAEL and LOAEL values could not be identified 14 

### by EPA from these studies due to inadequate reporting of exposure concentrati ons. 15 

### Rahman et al. (2007) conducted a cross-sectional st udy of workers at a urea fertilizer 16 

### factory in Bangladesh that consisted of an ammonia plant and a urea plant. The exposed group 17 

### studied consisted of 63 operators in the ammonia pl ant and 77 in the urea plant; 25 individuals 18 

### from the administration building served as a contro l group. Mean duration of employment 19 

### exceeded 16 years in all groups. Personal ammonia exposures were measured by two different 20 

### methods (Dräger PAC III and Dräger tube) in five to nine exposed workers per day for 10 21 

### morning shifts in the urea plant (for a total of 64 workers) and in five to nine exposed workers 22 

### per day for 4 morning shifts from the ammonia plant (for a total of 24 workers). Four to seven 23 

### volunteer workers per day were selected from the ad ministration building as controls for a total 24 

### of 25 workers over a five-day period. Questionnair es were administered to inquire about 25 

### demographics, past chronic respiratory disease, pas t and present occupational history, smoking 26 

### status, acute respiratory symptoms (cough, chest ti ghtness, runny nose, stuffy nose, and 27 

### sneezing), and use of protective devices. Lung fun ction tests (FVC, FEV 1 

### , and peak expiratory 28 

### flow rate [PEFR]) were administered preshift and po stshift (8-hour shifts) to the 88 exposed 29 

### workers after exclusion of workers who planned to h ave less than a four-hour working day; lung 30 

### function was not tested in the control group. Pers onal ammonia exposure and pulmonary 31 

### function were measured on the same shift for 28 exp osed workers. Linear multiple regression 32 

### was used to analyze the relationship between workpl ace and the percentage cross-shift change in 33



### FEV 1 

### ( ∆ FEV 1 

### %) while adjusting for current smoking. 34 

### Mean exposure levels at the ammonia plant determine d by the Dräger tube and Dräger 35 

### PAC III methods were 25.0 and 6.9 ppm (17.7 and 4.9 mg/m 3 

### ), respectively; the corresponding 36 

### means in the urea plant were 124.6 and 26.1 ppm (88 .1 and 18.5 mg/m 3 

### ) (Rahman et al., 2007). 37 

### Although the Dräger tube measurements indicated amm onia exposure about 4–5 times higher 38

### 24 DRAFT - DO NOT CITE OR QUOTE

than those obtained with the PAC III instrument, th ere was a significant correlation between the 1 

### ammonia concentrations measured by the two methods ( p = 0.001). No ammonia was detected 2 

### in the control area using the Dräger tube (concentr ations less than the measuring range of 2.5 to 3 

### 200 ppm [1.8 to 141 mg/m 3 

### ]). Based on an evaluation of the two monitoring m ethods and 4 

### communication with technical support at Dräger 5 

### , EPA considered the PAC III instrument to be 5 

### a more sensitive monitoring technology than the Drä ger tubes. Therefore, the PAC III air 6



### measurements were considered the more reliable meas urement of exposure to ammonia for the 7 

### Rahman et al. (2007) study. The study authors, how ever, observed that their measurements 8 

### indicated only relative differences in exposures be tween workers and production areas, and that 9 

### the validity of the exposure measures could not be evaluated based on their results. 10 

### The prevalence of acute respiratory symptoms was hi gher in the urea plant than in the 11 

### ammonia plant or in the administration building. C omparison between the urea plant and the 12 

### administration building showed that cough and chest tightness were statistically higher in the 13 

### former; a similar comparison of the ammonia plant a nd the administration building showed no 14 

### statistical difference in symptom prevalence betwee n the two groups (Table 4-4). Preshift 15 

### measurement of FVC, FEV 1 

### , and PEFR did not differ between urea plant and am monia plant 16 

### workers. Significant cross-shift reductions in FVC and FEV 1 

### were reported in the urea plant 17 

### (2 and 3%, respectively, p ≤ 0.05), but not in the ammonia plant. When control led for current 18 

### smoking, a significant decrease in ∆ FEV 1 

### % was observed in the urea plant ( p ≤ 0.05). Among 19



### 23 workers with concurrent measurements of ammonia and lung function on the same shift, 20 

### ammonia exposure was correlated with a cross-shift decline in FEV 1 

### of 3.9% per unit of log21 

### transformed ammonia concentration in ppm. EPA iden tified a NOAEL of 6.9 ppm (4.9 mg/m 3 

### ) 22



### and a LOAEL of 26.1 ppm (18.5 mg/m 3 

### ) in the Rahman et al. (2007) study based on increa sed 23 

### prevalence of respiratory symptoms and an acute dec rease in lung function. 24

25

5 Telephone conversations and e-mails dated June 22, 2010, from Michael Yanosky, Dräger Safety Inc., Tec hnical Support Detection Products to Amber Bacom, Syracuse Research Corporation [contractor to National Cente r for Environmental Assessment (NCEA), ORD, U.S. EPA].

### 25 DRAFT - DO NOT CITE OR QUOTE

Table 4-4. Prevalence of acute respiratory symptom s and cross-shift changes in lung function among workers exposed to a mmonia in a urea fertilizer factory

Parameter Ammonia plant (4.9 mg/m 3 ) a Urea plant (18.5 mg/m 3 ) a Administration building (concentration not determined) b

Respiratory symptoms Cough 4/24 (17%) c 18/64 (28%) d 2/25 (8%) Chest tightness 4/24 (17%) 21/64 (33%) d 2/25 (8%) Stuffy nose 3/24 (12%) 10/64 (16%) 1/25 (4%) Runny nose 1/24 (4%) 10/64 (16%) 1/25 (4%) Sneeze 0/24 (0%) 14/64 (22%) 2/25 (8%) Lung function parameters (cross-shift percentage ch ange) e , f

FVC 0.2 ± 9.3 -2.3 ± 8.8 ND

FEV 1 3.4 ± 13.3 -1.4 ± 8.9 ND

PEFR 2.9 ± 11.1 -1.0 ± 16.2 ND

ND: no data

a Mean ammonia concentrations measured by the Dräger PAC III method.

b Concentrations in the administration building were rejected by study authors due to relatively large d rift in the zero levels.

c Values are presented as incidence (prevalence expre ssed as a percentage).

d p ≤ 0.05 by Fisher’s exact test, comparing exposed wor kers to administrators.

e Calculated as ((Post shift-preshift)/preshift) x 10 0.

f Values are presented as mean ± SD.

Source: Rahman et al. (2007).

1 

### Tepper et al. (1991) evaluated pulmonary function ( FEV 1 

### ) changes among firefighters 2 

### from the city of Baltimore 6–10 years after a basel ine examination. The eligible study 3 

### population consisted of 963 firefighters of which 6 95 participated in the follow-up study. 4 

### Pulmonary function tests were performed for 628 fir efighters. Information about exposures was 5 

### obtained by questionnaire and by combining data fro m fire department records regarding the 6 

### number of fires fought by fire fighting units with individual work histories. To determine the 7 

### effects of occupational exposures while accounting for confounding by or interaction with other 8 

### risk factors, multiple linear regression techniques with dichotomous indicator variables were 9 

### used. Reported exposure to specific chemicals was rare, except for ammonia and chlorine; 10 

### 160 men reported exposure to ammonia and 128 to chl orine. Men with self-reported ammonia 11 

### exposure experienced a rate of decline in FEV 1 

### 1.7 times greater than men without ammonia 12 

### exposure, but the difference was not statistically significant. NOAEL and LOAEL values were 13 

### not identified in this study. 14

### 26 DRAFT - DO NOT CITE OR QUOTE

Hamid and El-Gazzar (1996) evaluated changes in ser um clinical chemistry as measures 1



### of neurochemical alterations and liver function amo ng workers at a urea production plant in 2 

### Alexandria, Egypt. The study group consisted of 60 male workers from the fertilizer plant, 3 

### including 30 workers with known exposures to ammoni a and 30 workers from the administrative 4 

### departments with no known history of exposure to am monia. The authors indicated that the 5 

### exposed population had worked at the fertilizer pla nt on average for 12 years. The exposed and 6 

### reference populations were matched on demographic c haracteristics including age, educational 7 

### status, and socioeconomic status. No information i s reported on exposure levels. Blood samples 8 

### were collected from each subject and analyzed for a spartate aminotransferase (AST), alanine 9 

### aminotransferase (ALT), hemoglobin, blood urea, and monoamine oxidase (MAO) and catalase 10 

### activity. Table 4-5 shows statistically significan t changes in hemoglobin and serum chemistry. 11 

### Mean levels of AST, ALT, and blood urea were signif icantly elevated among exposed workers 12 

### over controls. Mean levels of hemoglobin were sign ificantly lower, and MAO and catalase 13 

### enzyme activities were significantly depressed amon g exposed workers compared to controls. A 14 

### correlation analysis showed a positive correlation between catalase activity and levels of 15 

### hemoglobin, AST and ALT, and MAO activities. Hamid and El-Gazaar (1996) noted that 16



### inhibition of catalase can affect electrical stabil ity, permeability, and fluidity of membranes, 17 

### which may lead to hepatotoxic and neurotoxic altera tions in occupationally exposed workers. 18



### NOAEL and LOAEL values were not identified in this study due to the absence of information 19 

### on exposures at this fertilizer plant. 20

21 

### Table 4-5. Summary of significant changes in serum from workers

occupationally exposed to ammonia at a fertilizer p lant

Parameter Controls a Exposed a

ALT (U/mL) 16.0 ± 5.59 19.4 ± 5.69 b

AST (U/mL) 14.5 ± 4.67 17.9 ± 4.14 b

Hb (%) 14.8 ± 2.62 12.2 ± 2.29 c Blood urea (mg/mL) 0.203 ± 0.0512 0.319 ± 0.0755 c
MAO (units) 31.9 ± 10.1 20.8 ± 4.30 c

Catalase (IU/mL) 119.3 ± 4.76 80.9 ± 9.31 c

a Mean ± standard deviation.

b Significantly different from controls ( p < 0.05).

c Significantly different from controls ( p < 0.01).

Source: Hamid and El-Gazzar (1996).

22

### 27 DRAFT - DO NOT CITE OR QUOTE

4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIO ASSAYS IN 1



### ANIMALS—ORAL AND INHALATION 2 

### 4.2.1. Oral Exposure 3 

### Kawano et al. (1991) investigated the hypothesis th at the bacterium Helicobacter pylori 4 

### ( H. pylori ), which produces a potent urease that increases am monia production, plays a 5 

### significant role in the etiology of chronic atropic gastritis. Male Sprague Dawley rats (6/group) 6 

### were given tap water, 0.01%, or 0.1% ammonia ad lib itum for two or four weeks. The daily dose 7 

### of 0.01% and 0.1% ammonia in drinking water, based on a weight of 230 g for male rats and a 8 

### water consumption of 50 mL/day, was estimated to be 22 and 220 mg/kg-day, respectively. The 9



### effect of ammonia on the antral mucosa was estimate d by three measurements of the thickness of 10 

### the mucosa about 175 microns from the pyloric ring in the antral mucosa. The parietal cell 11 

### number per gland was determined at three locations in the oxyntic glandular area. Mucosal 12 

### lesions were not observed macro- or microscopically . There was a statistically significant 13



### decrease in mean antral mucosal thickness with incr easing dose and duration of exposure (Table 14 

### 4-6). Parietal cell number per oxyntic gland decre ased in a statistically significant dose- and 15 

### time-dependent fashion. The index of periodic acid -Schiff Alician blue positive intracellular 16 

### mucin was significantly lower in the antral and bod y mucosa with 0.1% ammonia; the index was 17 

### significantly lower only for the antral mucosa with 0.01% ammonia. The authors suggested that 18 

### administration of ammonia in drinking water causes gastric mucosal atrophy. Based on the 19 

### reduction in antral mucosal thickness, EPA identifi ed a LOAEL of 22 mg/kg-day; a NOAEL was 20



### not identified. 21

22 

### Table 4-6. Effect of ammonia in drinking water on t he thickness of the

gastric antral and body mucosa of the rat stomach

Length of treatment Thickness of mucosa (μM); mean ± sem

Control (tap water) Percent ammonia in drinking water 0.01% 0.1% Antral mucosa 2 weeks 270 ± 18 258 ± 22 217 ± 40 a
4 weeks 276 ± 39 171 ± 22 a 109 ± 12 b,c Body mucosa 2 weeks 574 ± 116 568 ± 159 591 ± 183 4 weeks 618 ± 154 484 ± 123 440 ± 80 a,c

a p < 0.05 vs. control group

b p < 0.01 vs. control group

c p < 0.01 versus 2 week treatment group

Source: Kawano et al. (1991).

23 

### In a follow-up study of the effect of ammonia produ ced from H. pylori , Tsujii et al. 24 

### (1993) studied the subchronic effect of ammonia in drinking water on the cell kinetics of the 25 

### gastric mucosa of the stomach. Six groups of Sprag ue Dawley male rats (36 rats/group) were 26

### 28 DRAFT - DO NOT CITE OR QUOTE

given 0.01% ammonia in drinking water for 3 days, o r 1, 2, 4 or 8 weeks; ammonia solutions 1 

### were changed daily. Tap water was provided for the balance of the 8-week study. A control 2 

### group was given tap water for eight weeks. Based o n the initial body weight (150 g) and 3 

### estimated daily water intake (50 mL), the daily dos e at a drinking water concentration of 0.01% 4 

### ammonia was estimated to be 33 mg/kg-day. Cellular migration was measured by labeling cells 5 

### with 5-bromo-2-deoxyuridine (BrDU) at different tim e periods and measuring the incorporation 6



### of this modified nucleoside with a histochemical te chnique using anti BrDU monoclonal 7 

### antibodies. Antral and body mucosa thickness was m easured as described in Kawano et al. 8 

### (1991). The measurement of cell proliferation in t he gastric mucosa was estimated using the 9 

### labeling index in gastric pits (ratio of labeled nu clei to total nuclei in the proliferation zone). T he 10 

### antral mucosal thickness decreased significantly at 4 and 8 weeks of treatment (Table 4-7) but 11 

### there was no effect on the body mucosa. Cell migra tion preceded the decrease in thickness of the 12 

### antral mucosa. The rate of cell migration (cells/d ay) toward the mucosal surface was 13 

### significantly greater for 0.01% ammonia-treated rat s compared to the control at 4 and 8 weeks of 14 

### treatment. Cell proliferation, as estimated from t he labeling index, was significantly increased 15 

### after one week for the antral and body mucosa. The authors concluded that 0.01% ammonia 16 

### increased epithelial cell migration in the antrum l eading to mucosal atrophy. The EPA identified 17 

### a LOAEL of 33 mg/kg-day based on decreased thicknes s of the gastric antrum; a NOAEL was 18



### not identified. 19

20 

### Table 4-7. Effect of ammonia in drinking water on g astric antral and body

mucosa in the stomach of Sprague-Dawley rats admini stered 0.01% ammonia in drinking water

Length of treatment Thickness of mucosa (μM) Antral mucosa Body mucosa Control (tap water only) 283 ± 26 534 ± 27 3 Days 305 ± 45 559 ± 50 1 Week 272 ± 31 542 ± 28 2 Weeks 299 ± 26 555 ± 37 4 Weeks 159 ± 29 b 531 ± 32 8 Weeks 168 ± 26 b 508 ± 29

a Extracted from Figure 3 of Tsujii et al. (1993); me an ± SD

b p < 0.05 vs. control (tap water only) group

Source: Tsujii et al. (1993)

21 

### Fazekas (1939) administered ammonium hydroxide to 5 1 rabbits (strain and sex not 22 

### specified) via gavage every other day initially and later daily in increasing amounts of 50–80 mL 23 

### as either a 0.5 or 1.0% solution over a long period of time. The daily dose (mg/kg-day) was 24 

### estimated using the weight of adult rabbits from st andard growth curve for rabbits (3.5–4.1 kg) 25 

### (U.S. EPA, 1988). Based on daily water consumption of 50–80 mL, a daily dose for the rabbits 26

### 29 DRAFT - DO NOT CITE OR QUOTE

receiving 0.5 and 1.0% ammonia in drinking water wa s approximately 61–110 mg/kg-day and 1 

### 120–230 mg/kg-day, respectively. The exact duratio n of the study is not reported, but it is clear 2 

### from the data that by the end of the experiment som e rabbits received only three or four doses 3 

### before dying as a result of intoxication in 5.5 day s and other rabbits received over 80 doses and 4 

### survived for up to 17 months. Toxicological endpoi nts evaluated included fluctuations in body 5 

### weights, changes in blood pressure measured at the central artery of the ear in 10 rabbits after 6 

### lengthy treatment, and changes in the weight, fat a nd cholesterol content of adrenals. For 7 

### comparison purposes, the weight of the adrenals fro m 41 healthy rabbits of similar age and body 8 

### weight were also determined. The average weight of adrenals from these 41 control rabbits was 9 

### 400.0 ± 13.4 mg. 10 

### Fazekas (1939) reported that differences in mean ad renal weight in ammonium 11 

### hydroxide-treated animals were significant, althoug h there was no description of the statistical 12 

### analysis performed in this study. Chemical evaluat ion of the adrenals from treated rabbits 13 

### revealed fat content 4.5 times greater and choleste rol content 6.5 times greater than controls. At 14 

### the beginning of the experiment, a greater weight l oss was observed among those rabbits 15 

### receiving ammonium hydroxide more frequently (daily ) at higher doses. Body weights 16 

### fluctuated among treated rabbits and generally decr eased initially and gradually increased in the 17 

### later months only to drop again a few weeks before death. Body weights for controls were not 18 

### reported. Thirteen rabbits exhibited weight increa ses after the initial loss that persisted until the 19 

### end of the experiment. Dissection of these rabbits revealed enlarged adrenals (800–1,340 mg), 20 

### and fatty tissue surrounding the kidneys, mesentery , and the pericardium. This fat accumulation 21 

### was not observed in untreated controls. Histology revealed enlarged cells of the zona fasciculata 22 

### of the adrenal cortex that were rich in lipid. The blood pressure of rabbits before dosing ranged 23 

### from 60 to 74 mm Hg and dropped with initial exposu re (during the first 5–10 minutes that lasted 24 

### up to 7 hours) to 20–30 Hg/mm. Following several m onths of ammonium hydroxide treatment, a 25 

### moderate elevation in blood pressure of 10–30 mm Hg was found in 8/10 rabbits. In the other 26 

### two rabbits, the blood pressure increased from the initial values of 62 and 65 to 90 Hg/mm 27 

### during the first 7 months of treatment and remained almost unchanged at this level until sacrifice. 28 

### In summary, Fazekas (1939) concluded that initial d ecreases in blood pressure and effects 29 

### of emaciation in rabbits following gavage treatment with ammonium hydroxide is associated 30 

### with the hypofunction of the cortical or medullary substance of the adrenal gland. The authors 31 

### also concluded that the subsequent increases in blo od pressure and body weight could be 32 

### attributed to hypertrophy of the adrenal cortex. T his study is limited by lack of reporting detail 33 

### and inadequate study design. The EPA did not ident ify a NOAEL or LOAEL from this study. 34 

### Toth (1972) evaluated whether hydrazine, methylhydr azines, and ammonium hydroxide 35 

### play a role in tumorigenesis in mice. Solutions of hydrazine (0.001%), methyl hydrazine 36 

### (0.01%), methyl hydrazine sulfate (0.001%), and amm onium hydroxide (0.1, 0.2, and 0.3%) 37 

### were administered continuously in the drinking wate r of 5- and 6-week-old randomly bred Swiss 38

### 30 DRAFT - DO NOT CITE OR QUOTE

mice (50/sex) for their entire lifetime. For ammon ium hydroxide, the study authors reported the 1 

### average daily drinking water intakes as 9.2, 8.2, a nd 6.5 mL/day for males for the 0.1, 0.2, and 2 

### 0.3% groups, respectively, and as 8.3, 6.5, and 4.8 mL/day for females, respectively. Given 3 

### these rates and assuming average default body weigh ts of 37.3 and 35.3 g for males and females, 4 

### respectively (U.S. EPA, 1988), the approximate cont inuous doses for ammonium hydroxide are 5 

### 250, 440, and 520 mg/kg-day for males and 240, 370, and 410 mg/kg-day for females. 6 

### Additionally, groups of C 3 

### H mice (40/sex) were exposed to ammonium hydroxide in the 7 

### drinking water at a concentration of 0.1% for their lifetime. Average daily water consumption 8 

### for these mice was reported as 7.9 and 8.4 mL/day f or males and females, respectively. The 9 

### approximate equivalent doses for these mice assumin g the same default body weights as above 10 

### (U.S. EPA, 1988) are 191 and 214 mg/kg-day for male s and females, respectively. Data were 11 

### not reported for a concurrent control group. Mice were monitored weekly for changes in body 12 

### weights and gross pathological changes were recorde d. The animals were either allowed to die 13 

### or were killed when found in poor condition. Compl ete necropsies were performed on all mice 14 

### and the liver, kidney, spleen, lung, and organs wit h gross lesions were processed for 15 

### histopathological examination. Data on body weight s were not reported. 16 

### For Swiss mice, tumor incidence at the 0.3% ammoniu m hydroxide concentration was as 17 

### follows: malignant lymphomas: 3/50 (males), 9/50 (f emales); and lung adenoma or 18 

### adenocarcinoma: 7/50 (male), 4/50 (female). Tumor incidence at the 0.2% ammonium 19 

### hydroxide concentration was: malignant lymphomas: 7 /50 (males), 10/50 (females); lung 20 

### adenoma or adenocarcinoma: 5/50 (male), 8/50 (femal e); and breast tumors: 4/50 (females). 21 

### Tumor incidence at the 0.1% ammonium hydroxide conc entration was: malignant lymphomas: 22 

### 4/50 (males), 10/50 (females); lung adenoma or aden ocarcinoma: 5/50 (male), 12/50 (female); 23 

### and breast tumors: 1/50 (females). The denominator s were not adjusted for survival, and 24 

### concurrent control data were not provided. For a s econd strain of mice (C 3 

### H) that received 0.1% 25 

### ammonium hydroxide in drinking water, the incidence of adenocarcinomas of the mammary 26 

### gland in female mice was 60%. The incidence of bre ast tumors in the corresponding untreated 27



### control mice was 76%. Other tumors were identified in treated rats, but were of low incidence. 28 

### Toth (1972) concluded that ammonium hydroxide was n ot carcinogenic in either strain of mouse. 29



### Because concurrent control tumor incidence was not provided other than the incidence of breast 30



### tumors in C 3 

### H female mice, the incidence of tumors in treated m ice cannot be independently 31 

### compared to control tumor incidence. 32 

### Tsujii et al. (1995, 1992a) evaluated the role of a mmonia in H. pylori -related gastric 33 

### carcinogenesis. H. pylori is a bacterium that produces a potent urease, whic h generates ammonia 34 

### from urea in the stomach, and has been implicated i n the development of gastric cancer. Tsujii et 35 

### al. (1995, 1992a) pretreated groups of 40–44 male S prague-Dawley rats with the initiator N36 

### methyl-N’-nitro-N-nitrosoguanidine (MNNG) in the dr inking water for 24 weeks before 37 

### administering 0.01% ammonium solution as a drinking fluid for 24 weeks. Based on an average 38

### 31 DRAFT - DO NOT CITE OR QUOTE

body weight of 523 g for male Sprague-Dawley rats d uring chronic exposure (U.S. EPA, 1988) 1 

### and a reported water consumption rate of 0.05 L/day , the approximate continuous dose 2 

### administered to these rats is 10 mg/kg-day. In eac h study, an additional group of 40–43 rats 3 

### given tap water for 24 weeks following pretreatment with MNNG served as controls. The study 4 

### protocol did not include a dose group that received ammonia only in drinking water. Stomachs 5 

### from rats surviving beyond 45 weeks were examined h istologically for evidence of ulcers, 6 

### lesions, and tumors. Tsujii et al. (1995) also eva luated serum gastrin levels from blood collected 7 

### at 30 and 46 weeks and mucosal cell proliferation i n animals surviving to 48 weeks by 8 

### calculating the labeling index (percentage ratio of labeled nuclei to total number of nuclei in the 9 

### proliferation zone) and the proliferation zone inde x (fraction of the gastric pit occupied by the 10 

### proliferation zone). 11 

### Tsujii et al. (1995, 1992a) observed a significantl y greater incidence of gastric cancers 12 

### among rats receiving ammonia after pretreatment wit h MNNG compared to rats receiving only 13 

### MNNG and tap water ( p < 0.01, χ

2 

### test). Seventy percent of MNNG+ammonia-treated ra ts 14 

### versus 31% of control rats developed gastric tumors in the first study (Tsujii et al., 1992a). The 15 

### number of gastric cancers per tumor-bearing rat in this study was 2.1 ± 1.4 among treated rats 16 

### and 1.3 ± 0.6 among control rats ( p < 0.01, χ

2 

### test). 17 

### In the second study, 66% of rats dosed with ammonia and pretreated with MNNG 18 

### developed gastric cancers compared to 30% of the co ntrol rats (Tsujii et al., 1995). The numbers 19 

### of gastric tumors per rat in this study were also s ignificantly higher among MNNG+ammonia20 

### exposed rats than controls ( p < 0.001, Mann-Whitney test) suggesting that ammoni a was a 21 

### promoter. In the absence of an ammonia-only treatm ent group, however, it is not possible to 22 

### distinguish with certainty between possible promoti on and initiator activity. The degree of 23



### differentiation of adenocarcinomas in control and a mmonia-treated rats was significantly 24



### different. Ammonia-treated rats also demonstrated a significantly higher incidence of larger 25 

### tumors (5.3 mm compared to 4.4 mm for controls) and of gastric cancers penetrating the 26 

### muscularis propria or deeper ( p < 0.01, 22% compared to controls 12%). In this st udy, the 27 

### labeling index and the proliferation zone index wer e statistically significantly elevated in 28 

### ammonia exposed rats compared to controls in the fu ndic mucosa and antral mucosa. 29 

### Tsujii et al. (1995) explored the hypothesis that a mmonia might increase intragastric pH, 30 

### leading to an increase in serum gastrin, a trophic hormone in the gastric fundus mucosa, and a 31 

### possible proliferating factor in gastric epithelial cells. The investigators found no significant 32 

### effects on serum gastrin levels and concluded that serum gastrin does not appear to play a 33 

### significant role in ammonia-induced promotion. 34

35 

### 4.2.2. Inhalation Exposure 36 

### Anderson et al. (1964) exposed a group of 10 guinea pigs (strain not given) and 10 Swiss 37 

### albino mice of both sexes continuously to 20 ppm (1 4 mg/m 3 

### ) ammonia vapors for up to 6 weeks 38

### 32 DRAFT - DO NOT CITE OR QUOTE

(anhydrous ammonia, purity not reported). Controls (number not specified) were maintained 1 

### under identical conditions except for the exposure to ammonia. An additional group of six 2 

### guinea pigs was exposed to 50 ppm (35 mg/m 3 

### ) for 6 weeks. The animals were observed daily 3 

### for abnormal signs or lesions. At termination, the mice and guinea pigs were sacrificed (two per 4 

### group at 1, 2, 3, 4, and 6 weeks of exposure) and s elected tissues (lungs, trachea, turbinates, 5 

### liver, and spleen) were examined for gross and micr oscopic pathological changes. No significant 6 

### effects were observed in animals exposed for up to 4 weeks, but exposure to 20 ppm (14 mg/m 3 

### ) 7 

### for 6 weeks caused darkening, edema, congestion, an d hemorrhage in the lung. Exposure of 8 

### guinea pigs to 50 ppm (35 mg/m 3 

### ) ammonia for 6 weeks caused grossly enlarged and c ongested 9 

### spleens, congested livers and lungs, and pulmonary edema. NOAEL and LOAEL values were 10 

### not identified in this study because only two anima ls per group were examined at a single time 11 

### period for the 20 ppm (14 mg/m 3 

### ) exposure groups (mice and guinea pigs). 12 

### Coon et al. (1970) exposed groups of male and femal e Sprague-Dawley and Long-Evans 13 

### rats, male and female Princeton-derived guinea pigs , male New Zealand rabbits, male squirrel 14 

### monkeys, and purebred male beagle dogs to 0, 155, o r 770 mg/m 3 

### ammonia (0, 219, or 15 

### 1,089 ppm) 8 hours/day, 5 days/week for 6 weeks (an hydrous ammonia, >99% pure). The 16 

### investigators stated that a typical loaded chamber contained 15 rats, 15 guinea pigs, 3 rabbits, 17 

### 3 monkeys, and 2 dogs. Blood samples were taken be fore and after the exposures for 18 

### determination of hemoglobin concentration, packed e rythrocyte volume, and total leukocyte 19 

### counts. Animals were routinely checked for clinica l signs of toxicity. At termination, sections 20 

### of the heart, lung, liver, kidney, and spleen were processed for microscopic examination in 21 

### approximately half of the surviving rats and guinea pigs and all of the surviving dogs and 22 

### monkeys. Sections of the brain, spinal cord, and a drenals from dogs and monkeys were also 23 

### retained, as were sections of the thyroid from the dogs. The nasal passages were not examined in 24 

### this study. 25 

### Exposure to 155 mg/m 3 

### ammonia did not result in any deaths or adverse cl inical signs of 26 

### toxicity in any of the animals. Hematological valu es were within normal limits for the laboratory 27 

### and there were no significant gross alterations in the organs examined. Microscopic examination 28 

### showed evidence of focal pneumonitis in the lung of one of three monkeys. Exposure to 770 29 

### mg/m 3 

### caused initial mild to moderate lacrimation and dy spnea in rabbits and dogs. However, 30 

### these clinical signs disappeared by the second week of exposure. No significant alterations were 31 

### observed in hematology tests or upon gross or micro scopic examinations at the highest dose. 32 

### However, consistent nonspecific inflammatory change s (not further described) were observed in 33 

### the lungs from rats and guinea pigs in the high-dos e group that were more extensive than in 34 

### control animals (incidence not reported). 35 

### Coon et al. (1970) also exposed rats (15–51/group) continuously to ammonia (anhydrous 36 

### ammonia, >99% pure) at 0, 40, 127, 262, 455, or 470 mg/m 3 

### (0, 57, 180, 371, 644, or 665 ppm) 37 

### for 90–114 days. Fifteen guinea pigs, three rabbit s, two dogs, and three monkeys were also 38

### 33 DRAFT - DO NOT CITE OR QUOTE

exposed continuously under similar conditions to am monia at 40 mg/m 3 

### (57 ppm) or 470 mg/m 3 1 

### (665 ppm). No significant effects were reported in any animals exposed to 40 mg/m 3 

### and 2 

### nonspecific inflammatory changes in the lungs and k idneys (not further described) were seen in 3 

### rats exposed to 127 mg/m 3 

### (180 ppm) ammonia. Exposure to 262 mg/m 3 

### (371 ppm) caused 4 

### nasal discharge in 25% of the rats and nonspecific circulatory and degenerative changes in the 5 

### lungs and kidneys (not further described, incidence not reported). A frank effect level at 6 

### 455 mg/m 3 

### (644 ppm) was observed due to high mortality in th e rats (50/51). Thirty-two of 7 

### 51 rats died by day 25 of exposure; no histopatholo gical examinations were conducted in these 8 

### rats. Exposure to 470 mg/m 3 

### (665 ppm) caused death in 13/15 rats and 4/15 guin ea pigs and 9 

### marked eye irritation in dogs and rabbits. Dogs experienced heavy lacrim ation and nasal 10 

### discharge, and corneal opacity was noted in rabbits . Hematological values did not differ 11 

### significantly from controls in animals exposed to 4 70 mg/m 3 

### (665 ppm) ammonia. 12 

### Histopathological evaluation of animals exposed to 470 mg/m 3 

### (665 ppm) consistently showed 13 

### focal or diffuse interstitial pneumonitis in all an imals and also alterations in the kidneys 14 

### (calcification and proliferation of tubular epithel ium), heart (myocardial fibrosis), and liver (fatty 15 

### change) in several animals of each species (inciden ce not reported). The study authors did not 16



### determine a NOAEL or LOAEL concentration from this study. EPA identified a NOAEL of 17 

### 40 mg/m 3 

### (57 ppm) and a LOAEL of 127 mg/m 3 

### (180 ppm) based on nonspecific inflammatory 18 

### changes in the lungs and kidneys in rats exposed to ammonia for 90 days. 19 

### Stombaugh et al. (1969) exposed groups of Duroc pig s (9/group) to measured 20 

### concentrations of 12, 61, 103, or 145 ppm ammonia ( 8, 43, 73, or 103 mg/m 3 

### ) continuously for 21 

### 5 weeks (anhydrous ammonia, purity not reported). Endpoints evaluated included clinical signs, 22 

### food consumption (measured 3 times/week), weight ga in (measured weekly), and gross and 23 

### microscopic examination of the respiratory tract at termination. A control group was not 24 

### included. In general, exposure to ammonia reduced food consumption and body weight gain, but 25 

### since a control group was not used, it is impossibl e to determine whether this reduction was 26 

### statistically significant. Food efficiency (food c onsumed/kg body weight gain) was not affected. 27 

### Exposure to ≥ 103 ppm (>73 mg/m 3 

### ) ammonia appeared to cause excessive nasal, lacrim al, and 28 

### mouth secretions and increased the frequency of cou gh (incidence data for these effects were not 29 

### reported). Examination of the respiratory tract di d not reveal any significant exposure-related 30 

### alterations. The study authors did not identify a NOAEL or LOAEL concentration from this 31 

### study. EPA did not identify a NOAEL or LOAEL value for this study due to the absence of a 32 

### control group. 33 

### Doig and Willoughby (1971) exposed groups of six sp ecific-pathogen-free derived 34 

### Yorkshire Landrace pigs to 0 or 100 ppm ammonia (0 or 71 mg/m 3 

### ) continuously for up to 35



### 6 weeks. The mean concentration of ammonia in the control chamber was 8 ppm (6 mg/m 3 

### ). 36 

### Additional groups of pigs were exposed to similar l evels of ammonia as well as to 0.3 mg/ft 3 

### of 37 

### ground corn dust to simulate conditions on commerci al farms. Pigs were monitored daily for 38

### 34 DRAFT - DO NOT CITE OR QUOTE

clinical signs and changes in behavior. Initial an d terminal body weights were measured to 1 

### determine body weight gain during the exposure peri od. Blood samples were collected prior to 2 

### the start of each experiment and at study terminati on for hematology (packed cell volume, white 3 

### blood cell, differential leukocyte percentage, and total serum lactate dehydrogenase). Two pigs 4 

### (one exposed and one control) were necropsied at we ekly intervals and tracheal swabs for 5 

### bacterial and fungal culture were taken. Histologi cal examination was conducted on tissue 6 

### samples from the lung, trachea, and bronchial lymph nodes. 7 

### During the first week of exposure, exposed pigs exh ibited slight signs of conjunctival 8 

### irritation including photophobia and excessive lacr imation. These irritation effects were not 9 

### apparent beyond the first week. Measured air conce ntrations in the exposure chambers increased 10 

### to more than 150 ppm (106 mg/m 3 

### ) on two occasions. Doig and Willoughby (1971) rep orted 11 

### that at this concentration, the signs of conjunctiv al irritation were more pronounced in all pigs. 12 

### No adverse effects on body weight gain were apparen t. Hematological parameters and gross 13 

### pathology were comparable between exposed and contr ol pigs. Histopathology revealed 14 

### epithelial thickening in the trachea of exposed pig s and a corresponding decrease in the numbers 15 

### of goblet cells as shown in Table 4-8. Tracheal th ickening was characterized by thinning and 16 

### irregularity of the ciliated brush border and an in creased number of cell layers. Changes in 17 

### bronchi and bronchioles characterized as lymphocyti c cuffing were comparable between exposed 18 

### and control pigs. Similarly, intraalveolar hemorrh age and lobular atelectasis were common 19 

### findings in both exposed and control pigs. Pigs ex posed to both ammonia and dust exhibited 20 

### similar reactions as those pigs exposed only to amm onia although initial signs of conjunctival 21 

### irritation were more severe in these pigs and these pigs demonstrated lesions in the nasal 22 

### epithelium similar to those observed in the trachea l epithelium of pigs exposed only to ammonia. 23

24 

### Table 4-8. Summary of histological changes observe d in rats exposed to

ammonia for 6 weeks

Duration of exposure (wks) Thickness of tracheal epithelium ( μ m) Number of tracheal goblet cells (per 500 μ m) Control 71 mg/m 3 NH 3 Control 71 mg/m 3 NH 3 1 15.7 21.0 13.6 24.0

2 20.4 29.3 22.7 10.3

3 20.4 36.6 18.9 7.3

4 21.8 36.2 18.3 10.7

5 19.3 33.2 20.2 10.0

6 18.9 41.6 20.0 1.3

Mean ± standard deviation 19.4 ± 2.1 32.9 ± 7.2 18.9 ± 3.0 10.6 ± 7.5

Source: Doig and Willoughby (1971).

25

### 35 DRAFT - DO NOT CITE OR QUOTE

Doig and Willoughby (1971) concluded that ammonia e xposure at 71 mg/m 3 

### may be 1 

### detrimental to young pigs. The authors suggested t hat although the structural damage to the 2 

### upper respiratory epithelium was slight, such chang es may cause severe functional impairment. 3 

### The study authors did not identify a NOAEL or LOAEL concentration from this study. EPA 4 

### identified a LOAEL of 71 mg/m 3 

### (100 ppm), based on damage to the upper respirator y 5 

### epithelium. A NOAEL could not be identified from t his single-concentration study. 6 

### Broderson et al. (1976) exposed groups of Sherman r ats (5/sex/dose) continuously to 7 

### 10 or 150 ppm ammonia (7 or 106 mg/m 3 

### , respectively) for 75 days (anhydrous ammonia, pur ity 8 

### not reported). The 10-ppm exposure level represent ed the background ammonia concentration 9 

### resulting from cage bedding that was changed 3 time s/week. The 150-ppm concentration 10 

### resulted from cage bedding that was replaced occasi onally, but never completely changed. F344 11 

### rats (6/sex/group) were exposed to ammonia in an in halation chamber at concentrations of 0 or 12 

### 250 ppm (177 mg/m 3 

### ) continuously for 35 days. Rats were sacrificed a t the end of the exposure 13 

### period and tissues were prepared for histopathologi cal examination of nasal passages, middle ear, 14 

### trachea, lungs, liver, kidneys, adrenal, pancreas, testicle, mediastinal lymph nodes, and spleen. 15 

### Histopathological changes were observed in the nasa l passage of rats exposed to 150 ppm 16 

### (106 mg/m 3 

### ) for 75 days (from bedding) or 250 ppm (177 mg/m 3 

### ) for 35 days (inhalation 17 

### chamber). Nasal lesions were most extensive in the anterior portions of the nose compared with 18 

### posterior sections of the nasal cavity. The respir atory and olfactory mucosa was similarly 19



### affected with a three- to fourfold increase in the thickness of the epithelium. Pyknotic nuclei and 20 

### eosinophilic cytoplasm were observed in epithelial cells located along the basement membrane. 21 

### Epithelial cell hyperplasia and formation of glandu lar crypts were observed and neutrophils were 22 

### located in the epithelial layer, the lumina of subm ucosal glands and the nasal passages. Dilation 23



### of small blood vessels and edema were observed in t he submucosa of affected areas. Collagen 24 

### replacement of submucosal glands and the presence o f lymphocytes and neutrophils were also 25 

### observed. No histopathological alterations were se en in control rats (10 ppm from bedding or 26 

### 0 ppm from the inhalation chamber). Broderson et a l. (1976) did not identify a NOAEL or 27 

### LOAEL from this study. EPA identified a NOAEL of 1 0 ppm (7 mg/m 3 

### ) and a LOAEL of 150 28 

### ppm (106 mg/m 3 

### ) based on nasal lesions in rats exposed to ammonia (from bedding) for 75 days. 29 

### Broderson et al. (1976) also studied the effect of ammonia inhalation on the incidence and 30 

### severity of murine respiratory plasmosis in mice in oculated with Mycoplasma pulmonis . These 31 

### results are presented in Section 4.4.3. 32 

### Gaafar et al. (1992) exposed 50 adult male white a lbino mice under unspecified 33 

### conditions to ammonia vapor derived from a 12% ammo nia solution (air concentrations were not 34 

### reported) 15 minutes/day, 6 days/week for up to 8 w eeks. Twenty-five additional mice served as 35 

### controls. Starting the fourth week, 10 exposed and five control mice were sacrificed weekly. 36 

### Following sacrifice, the nasal mucosa was removed a nd examined histologically. Frozen 37 

### sections of the nasal mucosa were subjected to hist ochemical analysis (succinic dehydrogenase, 38

### 36 DRAFT - DO NOT CITE OR QUOTE

nonspecific estrase, acid phosphatase, and alkaline phosphatase [ALP]). Histological 1 

### examination revealed a progression of changes in th e nasal mucosa from exposed rats from the 2 

### formation of crypts and irregular cell arrangements at 4 and 5 weeks, epithelial hyperplasia, 3 

### patches of squamous metaplasia, and loss of cilia a t 6 weeks, and dysplasia in the nasal 4 

### epithelium at 7 weeks. Similar changes were exagge rated in the nasal mucosa from rats 5 

### sacrificed at 8 weeks. Neoplastic changes included a carcinoma in situ in the nostril of one rat 6 

### sacrificed at 7 weeks that presented with loss of p olarity of the epithelium, hyperchromatism and 7 

### mitotic figures with an intact basement membrane, a nd an invasive adenocarcinoma in one rat 8



### sacrificed at 8 weeks. Histochemical results revea led changes in succinic dehydrogenase, acid 9 

### phosphatase, and ALP in exposed mice compared to co ntrols (magnitude of change not 10 

### reported), especially in areas of the epithelium ch aracterized by dysplasia. Succinic 11 

### dehydrogenase and acid phosphatase changes were lar gest in the superficial layer of the 12 

### epithelium, although the acid phosphatase reaction was stronger in the basal and intermediate 13 

### layers in areas of squamous metaplasia. ALP was st rongest in the goblet cells from the basal part 14 

### of the epithelium and basement membrane. 15 

### In summary, Gaafar et al. (1992) observed that amm onia exposure induces histological 16 

### changes in the nasal mucosa of male mice that incre ase in severity over longer exposure periods. 17 

### Corresponding abnormalities in histochemistry sugge st altered cell metabolism and energy 18 

### production, cell injury, cell proliferation, and po ssible chronic inflammation and neoplastic 19 

### transformation. The study authors did not determin e a NOAEL or LOAEL concentration from 20 

### this study. EPA did not identify a NOAEL or LOAEL because air concentrations were not 21 

### reported in the study. 22 

### Done et al. (2005) continuously exposed groups of 24 weaned pigs of several breeds in an 23 

### experimental facility to atmospheric ammonia at 0, 0.6, 10, 18.8, or 37 ppm (0, 0.4, 7, 13.3, or 26 24 

### mg/m 3 

### ) and 1.2, 2.7, 5.1, or 9.9 mg/m 3 

### inhalable dust for 5 weeks (16 treatment combinati ons). 25



### The concentrations of ammonia and dust used were re presentative of those found commercially. 26 

### A split-plot design was used in which one dust conc entration was allocated to a “batch” (which 27 

### involved five lots of 24 pigs each) and the four am monia concentrations were allocated to the 28



### four lots within that batch. The fifth lot served as a control. Each batch was replicated. In other 29 

### words, there were four dust concentrations × four a mmonia concentrations plus four controls 30 

### each replicated once giving 40 lots in total. In t otal, 960 pigs (460 males and 500 females) were 31 

### used in the study; 560 pigs were given postmortem e xaminations. Blood was collected from 15 32 

### sows before the start of the experiment and tested for porcine reproductive and respiratory 33 

### syndrome virus (PRRSV) and swine influenza. Five s entinel pigs were sacrificed at the start of 34 

### each batch and lung, nasal cavity, and trachea, tog ether with material from any lesions, were 35 

### examined postmortem and subjected to bacteriologica l examination. Postmortem examination 36 

### involved examining the pigs’ external surfaces for condition and abnormalities, examination of 37 

### the abdomen for peritonitis and lymph node size, in ternal gross examination of the stomach for 38

### 37 DRAFT - DO NOT CITE OR QUOTE

abnormalities, and gross examination of the nasal t urbinates, thorax, larynx, trachea, 1 

### tracheobronchial lymph nodes, and lung. Pigs were monitored for clinical signs (daily), growth 2 

### rate, feed consumption, and feed conversion efficie ncy (frequency of observations not specified). 3 

### After 37 days of exposure, eight pigs from each lot were sacrificed. Swabs of the nasal cavity 4 

### and trachea were taken immediately after death for microbiological analysis and the pigs were 5 

### grossly examined postmortem. On day 42, the remain ing pigs were removed from the exposure 6 

### facility and transferred to a naturally ventilated building for a recovery period of 2 weeks. Six 7 

### pigs from each lot were assessed for evidence of re covery and the remaining 10 pigs were 8 

### sacrificed and examined postmortem. 9 

### The pigs in this study demonstrated signs of respi ratory infection and disease common to 10 

### young pigs raised on a commercial farm (Done et al. , 2005). The different concentrations of 11 

### ammonia and dust did not have a significant effect on the pathological findings in pigs or on the 12 

### incidence of pathogens. In summary, exposure to am monia and inhalable dust at concentrations 13 

### commonly found at pig farms was not associated with increase in the incidence of respiratory or 14 

### other disease. The study authors did not identify a NOAEL or LOAEL concentration from this 15 

### study. EPA identified a NOAEL of 26 mg/m 3 

### (37 ppm), based on the lack of respiratory or othe r 16 

### disease following exposure to ammonia in the presen ce of respirable dust. 17 

### Weatherby (1952) exposed a group of 12 guinea pigs (strain not reported) to a target 18 

### concentration of 170 ppm (120 mg/m 3 

### ) 6 hours/day, 5 days/week for up to 18 weeks (anhy drous 19 

### ammonia, purity not reported). The actual concentr ation measured in the exposure chamber 20 

### varied between 140 (99 mg/m 3 

### ) and 200 ppm (141 mg/m 3 

### ). A control group of six guinea pigs 21 

### was exposed to room air. All animals were weighed weekly. Interim sacrifices were conducted 22 

### at intervals of 6 weeks (four exposed and two contr ol guinea pigs) and the heart, lungs, liver, 23 

### stomach and small intestine, spleen, kidneys, and a drenal glands were removed for microscopic 24 

### examination; the upper respiratory tract was not ex amined. No exposure-related effects were 25 

### observed in guinea pigs sacrificed after 6 or 12 we eks of exposure. However, guinea pigs 26 

### exposed to ammonia for 18 weeks showed considerable congestion of the spleen, liver, and 27 

### kidneys, and early degenerative changes in the adre nal gland. The most severe changes occurred 28 

### in the spleen and the least severe changes occurred in the liver. The spleen of exposed guinea 29 

### pigs contained a large amount of hemosiderin and ki dney tubules showed cloudy swelling with 30 

### precipitated albumin in the lumens and some urinary casts (cylindrical structures indicative of 31 

### disease). The incidence of histopathological lesio ns was not reported. EPA identified the 32 

### ammonia concentration of 170 ppm (120 mg/m 3 

### ) to be a LOAEL based on congestion of the 33 

### spleen, liver, and kidneys and early degenerative c hanges in the adrenal gland. A NOAEL could 34 

### not be identified in this single-concentration stud y. 35 

### Curtis et al. (1975) exposed groups of crossbred p igs (4–8/group) to 0, 50, or 75 ppm 36 

### ammonia (0, 35, or 53 mg/m 3 

### ) continuously for up to 109 days (anhydrous ammoni a, >99.9% 37 

### pure). Endpoints evaluated included clinical signs and body weight gain. At termination, all 38

### 38 DRAFT - DO NOT CITE OR QUOTE

pigs were subjected to a complete gross examination and representative tissues from the 1 

### respiratory tract, the eye and its associated struc tures, and the visceral organs (not specified) 2 

### were taken for subsequent microscopic examination. Weight gain was not significantly affected 3 

### by exposure to ammonia and the results of the evalu ations of tissues and organs were 4 

### unremarkable. The turbinates, trachea, and lungs o f all pigs were classified as normal. The 5 

### study authors did not identify a NOAEL or LOAEL fro m this study. EPA identified a NOAEL 6 

### of 75 ppm (53 mg/m 3 

### ) based on the absence of effects occurring in pigs exposed to ammonia; a 7 

### LOAEL was not identified from this study. 8

9



### 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND I NHALATION 10 

### Diekman et al. (1993) reared 80 crossbred gilts (y oung female pigs) in a conventional 11 

### grower from 2 to 4.5 months of age; pigs were expos ed naturally during that time to Mycoplasma 12 

### hypopneumoniae and Pasteurella multocida , which causes pneumonia and atrophic rhinitis, 13 

### respectively. At 4.5 months of age, the pigs were transferred to environmentally regulated rooms 14 

### where they were exposed continuously to a mean conc entration of ammonia of 7 ppm (range, 4– 15 

### 12 ppm) (5 mg/m 3 

### ; range, 3–8.5 mg/m 3 

### ) or 35 ppm (range, 26–45 ppm) (25 mg/m 3 

### ; range, 18–32 16 

### mg/m 3 

### ) for 6 weeks (Diekman et al., 1993). A control gr oup was not included in this study. The 17 

### low concentration of ammonia was obtained by the fl ushing of manure pits weekly and the 18 

### higher concentration of ammonia was maintained by a dding anhydrous ammonia (purity not 19 

### reported) to manure pits that were not flushed. Af ter 6 weeks of exposure, 20 gilts from each 20 

### group were sacrificed and sections of the lungs and snout were examined for gross lesions. In 21 

### addition, the ovaries, uterus, and adrenal glands w ere weighed. The remaining 20 gilts/group 22 

### were mated with mature boars and continued being ex posed to ammonia until gestation day 30, 23 

### at which time they were sacrificed. Fetuses were e xamined for viability, weight, and length and 24 

### the number of copora lutea were counted. 25 

### Gilts exposed to 35 ppm (25 mg/m 3 

### ) ammonia gained less weight than gilts exposed to 26 

### 7 ppm (5 mg/m 3 

### ) during the first 2 weeks of exposure (7% decrease

, p < 0.01), but growth rate 27



### recovered thereafter. Mean scores for lesions in t he lungs and snout were not statistically 28 

### different between the two exposure groups and there were no differences in the weight of the 29 

### ovaries, uterus, and adrenals. Age at puberty did not differ significantly between the two groups, 30 

### but gilts exposed to 35 ppm (25 mg/m 3 

### ) ammonia weighed 7% less ( p < 0.05) at puberty than 31 

### those exposed to 7 ppm (5 mg/m 3 

### ). In gilts that were mated, conception rates were similar 32 

### between the two groups (94.1 vs. 100% in low vs. hi gh exposure). At sacrifice on day 30 of 33 

### gestation, body weights were not significantly diff erent between the two groups. In addition, 34 

### there were no significant differences between the t wo groups regarding percentage lung tissue 35 

### with lesions and mean snout grade. Number of corpo ra lutea, number of live fetuses, and weight 36 

### and length of the fetuses on day 30 of gestation we re not significantly different between 37 

### treatment groups. Diekman et al. (1993) did not id entify NOAEL or LOAEL concentrations for 38

### 39 DRAFT - DO NOT CITE OR QUOTE

maternal or fetal effects in this study. The EPA d id not identify NOAEL or LOAEL values from 1 

### this study due to the absence of a control group an d due to confounding by exposure to bacterial 2 

### and mycoplasm pathogens. 3

4



### 4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES 5



### 4.4.1. Acute Oral Studies 6 

### Two acute oral studies have been conducted using am monia. Gastrointestinal effects 7 

### were observed in rats following gavage with ammoniu m hydroxide (Nagy et al., 1996; Takeuchi 8 

### et al., 1995). Takeuchi et al. (1995) reported hem orrhagic necrosis of the gastric mucosa in male 9 

### Sprague-Dawley rats administered ammonium hydroxide once (by gavage) at ≥ 1% ammonium 10 

### hydroxide (dose could not be estimated because gava ge volume was not reported). Nagy et al. 11 

### (1996) observed severe hemorrhagic mucosal lesions in female Sprague-Dawley rats (6/group) 12 

### 15 minutes after exposure to 48 mg/kg ammonium hydr oxide via gavage (dose estimated by EPA 13 

### assuming an average body weight of 0.185 kg). 14 

### Leakage of cysteine proteases into the gastric lume n occurred 15 minutes after exposure 15 

### and gastrointestinal effects were prevented by pret reatment with a sulfhydryl alkylating 16 

### compound (N-ethylmaleimide) (Nagy et al., 1996). 17

18



### 4.4.2. Acute and Short-Term Inhalation Studies 19 

### Several acute and short-term inhalation studies (ex posure duration of ≤ 30 days) have 20 

### been conducted using ammonia. The lethality of amm onia inhalation has been investigated in 21 

### rats and mice (rat LC 50 

### values ≥ 11,590 mg/m 3 

### ; mouse LC 50 

### values ≥ 2,990 mg/m 3 

### ) (Appleman et 22 

### al., 1982; Kapeghian et al., 1982; Hilado et al., 1 977; Silver and McGrath, 1948). Clinical signs 23



### of acute ammonia inhalation in rats and mice includ ed eye and nose irritation, dyspnea, ataxia, 24 

### seizures, coma, and death (Appleman et al., 1982; K apeghian et al., 1982). Decreased body 25 

### weight gain was observed in rats, mice, and pigs to ammonia concentrations as low as 400 26 

### mg/m 3 

### (500 ppm) for up to 30 days (Gustin et al., 1994; Urbain et al., 1994; Kapeghian et al., 27 

### 1982; Richard et al., 1978a). 28 

### Ammonia inhalation produced changes in pulmonary fu nction, with decreased respiratory 29



### rate observed in rats at concentrations ≥ 848 mg/m 3 

### (1,200 ppm) (Rejniuk et al., 2008, 2007), in 30



### mice at concentrations ≥ 214 mg/m 3 

### (303 ppm) (Kane et al., 1979), and in rabbits at 31



### concentrations ≥ 35 mg/m 3 

### (50 ppm) (Mayan and Merilan, 1972). Decreased air way pressure and 32 

### pO 2 

### were also observed in rabbits exposed to ammonia c oncentrations ≥ 24,745 mg/m 3 33 

### (35,000 ppm) for 4 minutes (Sjöblom et al., 1999). Histopathological changes in the respiratory 34 

### tract of ammonia-exposed rats ( ≥ 141 mg/m 3 

### ; 200 ppm) and mice ( ≥ 216 mg/m 3 

### ; 305 ppm) 35 

### included alterations in the nasal and tracheal epit helium (i.e., irritation and inflammation) and 36 

### pneumonitis, atelectasis, and intralveolar hemorrha ge of the lower lung (Buckley et al., 1984; 37 

### Kapeghian et al., 1982; Richard et al., 1978a; Gamb le and Clough, 1976). Dodd and Gross 38

### 40 DRAFT - DO NOT CITE OR QUOTE

(1980) showed that pulmonary function deficits meas ured 1, 7, 21, and 35 days postexposure 1 

### were correlated with lung histopathology in cats ex posed to ammonia (707 mg/m 3 

### or 1,000 ppm 2 

### for 10 minutes). Acute effects were followed by ch ronic respiratory dysfunction that was 3 

### characterized by secondary bronchitis, bronchioliti s, and bronchopneumonia. 4



### Several studies evaluated cardiovascular and/or met abolic effects of acute or short-term 5 

### ammonia exposure. Bradycardia was observed in rabb its exposed to 1,768 mg/m 3 

### (2,500 ppm) 6 

### for 180 minutes and arterial pressure variations we re seen at 3,535 mg/m 3 

### (5,000 ppm) (Richard 7 

### et al., 1978b). Acidosis, as evidenced by a decrea se in blood pH and an increase in arterial blood 8 

### pCO 2 

### , occurred in rats exposed to 212 mg/m 3 

### (300 ppm) ammonia for 5, 10, or 15 days 9 

### (Manninen et al., 1988) and in rabbits exposed to ≥ 3,535 mg/m 3 

### (5,000 ppm) for up to 10 

### 180 minutes (Richard et al., 1978b). No effects on blood pH, pO 2 

### , or pCO 2 

### were seen in rats 11 

### exposed to ≤ 818 mg/m 3 

### (1,157 ppm) (Schaerdel et al., 1983) or cattle exp osed to concentrations 12 

### of <71 mg/m 3 

### (100 ppm) (Mayan and Merilan, 1976). Several stud ies also investigated amino 13



### acid levels and neurotransmitter metabolism in the brain following acute inhalation exposure to 14 

### ammonia in rats and mice (Manninen and Savolainen, 1989; Manninen et al., 1988; Sadasivudu 15 

### et al., 1979; Sadasivudu and Murthy, 1978). It has been suggested that glutamate and γ -amino 16 

### butyric acid (GABA) play a role in ammonia-induced neurotoxicity. These acute and short-term 17 

### studies are further described in Appendix C.5. 18

19 

### 4.4.3. Immunotoxicity 20 

### Secondary infection has been observed in humans who have received severe burns from 21 

### exposure to highly concentrated aerosols derived fr om ammonia (Sobonya, 1977; Taplin et al., 22 

### 1976). However, there is no evidence that the decr eased resistance to infection in these cases 23 

### represents a primary impairment of the immune syste m in humans since necrosis of exposed 24 

### tissues facilitates infection by pathogenic organis ms. Nevertheless, animal studies have shown 25 

### that exposure to airborne ammonia may impair immune function as described below (Gustin et 26 

### al., 1994; Neumann et al., 1987; Targowski et al., 1984; Schoeb et al., 1982; Richard et al., 27 

### 1978a; Broderson et al., 1976). 28 

### Broderson et al. (1976) exposed Sherman and F344 r ats (11–12/sex/dose) continuously to 29 

### ammonia concentrations of 108 or 212 ppm ammonia (7 6 or 150 mg/m 3 

### ), respectively (from 30 

### soiled bedding), for 7 days prior to inoculation wi th M. pulmonis and for 30–35 days following 31 

### inoculation. The matched control groups (bedding c hanged daily) were exposed to 11 ppm 32 

### (8 mg/m 3 

### , Sherman rats) or 2 ppm (1 mg/m 3 

### , F344 rats). Additional groups of F344 rats 33 

### (6/sex/group) were exposed continuously to 25 (two groups), 50, 100, or 250 ppm ammonia (18, 34 

### 35, 71, or 177 mg/m 3 

### , respectively) in an inhalation chamber for 7 days prior to inoculation with 35 

### M. pulmonis and for up to 42 days following inoculation. Each treatment group had a 36 

### corresponding control group that was inoculated wit h M. pulmonis in order to produce murine 37 

### respiratory mycoplasmosis (MRM). Clinical observat ions were performed (frequency not 38

### 41 DRAFT - DO NOT CITE OR QUOTE

given). Rats were sacrificed at the end of the exp osure period and tissues were prepared for 1 

### histopathological examination of nasal passages, mi ddle ear, trachea, lungs, liver, kidneys, 2 

### adrenal, pancreas, testicle, spleen, mediastinal ly mph nodes, and spleen. 3 

### Typical signs of MRM (i.e., snuffling, head shakin g) began in all rat groups 4 

### approximately 10 days after inoculation. Rats expo sed to ammonia exhibited dyspnea and 5 

### hyperpnea, had rough coats, and sat in a hunched po sture. All ammonia concentrations 6 

### significantly increased the severity of rhinitis, o titis media, tracheitis, and pneumonia 7



### characteristic of M. pulmonis . Furthermore, there was a significant concentrati on response 8 

### between observed respiratory lesions and increasing environmental ammonia concentration for 9 

### gross and microscopic lesions (see Table 4-9). All lesions observed were characteristic of MRM. 10 

### Gross bronchiectasis and/or pulmonary abscesses and the extent of gross atelectasis and 11 

### consolidation were consistently more prevalent in e xposed animals at all ammonia 12 

### concentrations than in their corresponding controls . Increasing ammonia concentration was 13 

### associated with an increasing rate of isolating of M. pulmonis from the respiratory tract. EPA 14 

### identified a LOAEL of 18 mg/m 3 

### (25 ppm) for increased severity of rhinitis, otiti s media, 15 

### tracheitis, and pneumonia, and increased incidence of respiratory lesions in F344 rats. A 16 

### NOAEL was not identified in this study. 17

18 

### Table 4-9. Incidence of pulmonary lesions in rats inoculated with

M. pulmonis and exposed to ammonia (7 days later for 28–42 day s)

Ammonia concentration (mg/m 3 ) Incidence of gross lesions (%) Incidence of microsc opic lesions (%)

Ammonia Matched control Ammonia Matched control

Soiled bedding 76 20/24 (83%) a 10/23 (43%) 23/24 (96%) a 14/23 (61%) 150 6/22 (27%) 2/24 (8%) 19/22 (86%) a 8/24 (33%)

Inhalation chamber b

18 5/12 (42%) 1/12 (8%) 9/12 (75%) 6/12 (50%) 18 6/12 (50%) 4/12 (33%) 9/12 (75%) 7/12 (58%) 40 8/12 (66%) 2/12 (17%) 10/12 (83%) 9/12 (75%) 70 4/12 (33%) 1/12 (8%) 12/12 (100%) 6/12 (50%) 180 10/12 (83%) 2/12 (17%) 12/12 (100%) 2/12 (17%)

a p ≤ 0.01, χ 2 test comparing ammonia-exposed group to matched co ntrols.

b p ≤ 0.01, χ 2 test comparing the sum of all ammonia group to the sum of matched controls. Regression analysis showed that increasing concentrations of ammonia we re related to the increased incidence of gross and microscopic lesions.

Source: Broderson et al. (1976).

19 

### Richard et al. (1978a) exposed groups of 99 male O FI mice continuously to ammonia 20 

### concentrations of 0 or 500 ppm (0 or 354 mg/m 3 

### ) for 8 hours and for 168 hours prior to infection 21

### 42 DRAFT - DO NOT CITE OR QUOTE

by aerosolized P. multocida (2-minute exposure to a predetermined 50% lethal c oncentration of 1 

### bacteria). Mice were observed for 12 days for mort ality. At 168 hours, a significant increase in 2 

### mortality was observed among ammonia-exposed mice c ompared to controls (+36%) 3 

### (Table 4-10). Based on supporting findings in rat studies conducted by these same researchers 4 

### (summarized under Section 4.4.2), Richard et al. (1 978) suggested that the irritating action of 5 

### ammonia destroys the tracheobronchial mucosa and ca uses inflammatory lesions so that the 6 

### sensitivity to respiratory infection increased with exposure to ammonia for 168 hours, while the 7 

### same exposure for 8 hours was not sufficient to inc rease this sensitivity. 8

9 

### Table 4-10. Mortality in P. multocida -infected mice exposed to ammonia for

8 or 168 hours

Exposure duration Controls Exposed (400 mg/m 3 ) 8 h 23/49 (47%) a 21/49 (43%) 168 h 25/50 (50%) 42/49 (86%) b

a Number of deaths/total number of animals (percentag e mortality).

b Significantly different from controls ( p < 0.05, Student’s t-test).

Source: Richard et al. (1978a).

10 

### Schoeb et al. (1982) exposed pathogen-free F344 rat s (5–15 rats/dose) continuously to 11



### ammonia concentrations of ≤ 2 ppm ( ≤ 1 mg/m 3 

### ; controls, air concentration was usually not 12 

### measureable and never exceeded 2 ppm) or 100 ppm (7 1 mg/m 3 

### ) for either 7 days prior to 13



### inoculation with M. pulmonis , or simultaneously with inoculation, for up to 28 days. Rats were 14 

### sacrificed in groups of five at unspecified interva ls up to 28 days following inoculation. At 15 

### sacrifice, serum was collected from each rat for de termination of IgG and IgM antibodies against 16 

### M. pulmonis by enzyme-linked immunosorbent assay. Nasal passa ges, larynges, tracheas, and 17 

### lungs were collected for quantitative assessment of M. pulmonis growth. Growth of M. pulmonis 18 

### increased most rapidly in the nasal passages and su bsequently in the larynx, trachea, and lungs in 19 

### both control and exposed rats. However, overall gr owth of M. pulmonis was much greater in 20 

### ammonia-exposed rats than controls. Serum immunogl obin antibody responses (IgG and IgM) 21 

### were also greater in ammonia-exposed rats than in c ontrols. Schoeb et al. (1982) also conducted 22 

### two additional in vivo experiments to evaluate pulm onary absorption and clearance of ammonia 23 

### from the respiratory tract and one in vitro experim ent to evaluate whether increased populations 24



### of M. pulmonis in ammonia-exposed rats are the result of direct e nhancement of growth of the 25 

### organism or indirect effects on the host. In one i n vivo experiment, 11 rats that were exposed in 26 

### the first experiment to ammonia at 100 ppm (71 mg/m 3 

### ) for 1 week were exposed under 27



### anesthesia to ammonia via face mask at ≤ 500 ppm (354 mg/m 3 

### ) for an unspecified length of 28 

### time. Tracheas were surgically exposed, cannulated , and attached to a gas detector for 29



### measurement of ammonia concentration in the trachea . Ammonia was not detected in the 30

### 43 DRAFT - DO NOT CITE OR QUOTE

tracheal air of any rats during exposure to <500 pp m (<354 mg/m 3 

### ), and measured 10–20 ppm 1 

### (7–14 mg/m 3 

### ) in some rats (number not specified) at 500 ppm (3 54 mg/m 3 

### ). These results 2 

### indicate that virtually all ammonia is absorbed in the nasal passages in rats exposed to <500 ppm 3 

### (<354 mg/m 3 

### ) ammonia. In the other in vivo experiment, groups of 38 F344 rats were exposed 4 

### to either 0 or 100 ppm (0 or 71 mg/m 3 

### ) ammonia for 1 week prior to inoculation with 5



### radiolabeled Staphylococcus epidermis (a bacterium commonly present on human skin) to 6 

### measure pulmonary bacterial clearance. Following i noculation, half of the exposed animals and 7 

### half of the controls were sacrificed immediately an d the remaining rats were sacrificed 6 hours 8 

### later. There were no significant differences betwe en ammonia-exposed and control rats 9 

### regarding the rate of pulmonary clearance (combined results showed 91 ± 6% clearance of 10 

### bacteria for both groups in 6 hours). In vitro, gr owth of M. pulmonis was inhibited by 1 mM 11



### ammonium ion added to a culture medium as ammonium hydroxide. Lower concentrations 12 

### (0.01, 0.1, and 0.5 mM) had no effect. The study a uthors suggest that these data indicate that 13 

### ammonia increases growth of M. pulmonis indirectly through effects on the host. 14 

### Hartley guinea pigs (8/concentration, sex not spec ified) vaccinated with Mycobacterium 15



### bovis BCG and challenged intradermally with 2.5 μg of pu rified protein derivative (PPD) of 16 

### tuberculin were continuously exposed to ammonia con centrations of 50 or 90 ppm (35 or 17 

### 64 mg/m 3 

### ) for 3 weeks (Targowski et al., 1984). An additio nal eight PPD-positive guinea pigs 18 

### exposed to a normal environment with ammonia concen trations <15 ppm (<11 mg/m 3 

### ) served as 19 

### controls. Guinea pigs were again challenged intrad ermally with PPD following the exposure 20 

### period. Clinical observations were recorded daily and body weights were measured at the 21 

### beginning and end of the experiment. Blood samples collected at the beginning and end of the 22 

### experiment were submitted for determination of ammo nia content and hematology (red blood 23 

### cells and white blood cells). Cultures of separate d blood lymphocytes and bronchial leukocytes 24 

### were evaluated for responsiveness to mitogens (1 μ g/mL phytohemagglutin [PHA] or 25 

### concanavalin A) or 24 μg/mL PPD. Cultures of alveo lar and bronchial macrophages from 26 

### control and exposed guinea pigs were measured for b actericidal and phagocytic capacities in the 27 

### presence of Staphylococcus aureus . Targowski et al. (1984) also evaluated the effec t of 28 

### ammonia exposure on the in vitro immune response in lymphocytes and macrophages collected 29 

### from five normal Hartley guinea pigs. The percenta ge of viable lymphocytes (unstained) was 30 

### determined by trypan blue exclusion test at 0, 2, 4 , 24, 48, and 72 hours following incubation in 31 

### medium alone or medium containing ammonia concentra tions ranging from 1 to 100 mg/L 32 

### (ammonium hydroxide added to medium). Macrophages were incubated with concentrations of 33 

### ammonia ranging from 0.1 to 50 mg/L for 1 hour prio r to the addition of S. aureus

. 34 

### Guinea pigs exposed to ammonia at 50 or 90 ppm (35 or 64 mg/m 3 

### ) did not exhibit any 35 

### significant changes in body weight and did not show any notable signs of distress, conjunctivitis, 36 

### or respiratory disease (Targowski et al., 1984). D ermal response to PPD measured as the 37 

### diameter of erythema was significantly less at p < 0.05 (Student’s t-test) in guinea pigs exposed 38

### 44 DRAFT - DO NOT CITE OR QUOTE

to 64 mg/m 3 

### at 24, 72, and 96 hours compared to controls (Tabl e 4-11). No significant 1 

### hematological changes were observed between exposed and control groups. The response to 2 

### mitogens and PPD of blood lymphocytes and bronchial lymphocytes from animals exposed to 90 3 

### ppm (64 mg/m 3 

### ) ammonia was significantly less than the response fr om control animals ( p < 4 

### 0.01) (shown graphically by authors). The lymphocy te response for guinea pigs exposed to 50 5 

### ppm (35 mg/m 3 

### ) ammonia was similar to control. In vitro exposur e of blood lymphocytes to 6 

### ammonia at 10 or 100 mg/L resulted in decreased via bility and reduced response to PHA 7 

### stimulation (significantly different from controls at p < 0.01). Lower concentrations of ammonia 8 

### ( ≤ 1 mg/L) did not significantly affect viability or s uppress the responsiveness of lymphocytes in 9 

### vitro. Bactericidal and phagocytic activity of alv eolar macrophages from animals exposed to 10 

### ammonia was not significantly affected. Similarly, phagocytic activity was not significantly 11 

### affected in vitro following treatment with ammonia at 1–100 mg/L. However, a significant 12 

### inhibition in the macrophage bactericidal activity was observed in vitro at 0.01 and 0.05 mg/L ( p 13 

### < 0.01). No significant changes in bactericidal ac tivity were observed in cultures treated with 14



### ammonia at ≤ 1 mg/L. 15

16 

### Table 4-11. Dermal response to the injection of tu berculin in animals

exposed to ammonia for 3 weeks (mean diameter of re dness in mm)

Time of observation (hrs) Ammonia concentration (mg/m 3 )

Control 35 64

24 17.6 ± 1.7 a 15.2 ± 2.23 14.4 ± 2.2 b

48 15.5 ± 1.9 13.8 ± 1.6 14.7 ± 1.8

72 12.0 ± 1.1 12.6 ± 1.0 8.7 ± 1.4 b

96 10.4 ± 1.5 12.6 ± 0.7 0.0 b

a Mean ± standard deviation.

b Significantly different from controls ( p < 0.05, Student’s t-test).

Source: Targowski et al. (1984).

17 

### Neumann et al. (1987) conducted three experiments to evaluate the ability of unweaned 18 

### piglets exposed to ammonia to fight off infection f ollowing an intranasal challenge with P. 19



### multocida (Carter type A). In the first experiment (Trial 1 ), unweaned piglets were exposed to 20 

### ammonia at 50 ppm (35 mg/m 3 

### ) for 42 days either without additional treatment ( 22 piglets) or 21 

### following intranasal challenge with P. multocida (10 piglets). Groups of 8–10 piglets were also 22 

### exposed for 35 days during the second experiment (T rial 2) under these same conditions. A 23 

### thermo-motor stress swim test (water temperature wa s 15°C) was also included as an additional 24 

### condition during Trial 2. During the third experim ent (Trial 3), all three conditions (i.e., 25 

### ammonia inhalation, intranasal challenge with P. multocida and a thermo motor swim test) were 26 

### tested in groups of 8–10 piglets; however, the pigl ets were exposed to 100 ppm (71 mg/m 3 

### ) 27

### 45 DRAFT - DO NOT CITE OR QUOTE

ammonia for 31 days. The test groups were compared to corresponding controls during each 1 

### trial. Blood samples were collected on multiple da ys during each trial and analyzed for nucleolar 2 

### activity of peripheral lymphocytes and determinatio n of gamma globulins. Lavage fluid from the 3 

### lungs was collected on multiple days to characteriz e differentiation of bronchoalveolar cells. 4 

### Additionally, on the 23 rd 

### day of exposure, a small test group (five test and five control pigs from 5 

### Trial 2) was exposed to a radioactive aerosol (Tc 99m 

### -tagged sulfur colloid complex suspended in 6 

### NaCl) through a breathing mask for 10 minutes to ev aluate pulmonary aerosol distribution 7 

### patterns. During this test, the dorsal pulmonary r adioactivity was recorded using a gamma 8



### camera. 9 

### Lymphocyte nucleolar activity increased in a dose-r elated manner following ammonia 10 

### exposure (data not shown) (Neumann et al., 1987). A more pronounced increase was observed 11 

### in the presence of infection and was further intens ified under additional thermo-motor stress. 12 

### Neumann et al. (1987) suggested that ammonia concen trations of 35 and 71 mg/m 3 

### can cause 13 

### activation of RNA synthesis in the cell nucleus of the peripheral lymphocytes of piglets, as 14 

### evidenced by an increase in the percentage of lymph ocytes with compact nucleoli. Gamma 15 

### globulin concentrations were not significantly affe cted at 35 mg/m 3 

### ammonia, but in pigs 16 

### inhaling 71 mg/m 3 

### ammonia, gamma globulin concentrations were signif icantly lower than 17 

### controls ( α = 0.01, Wilcoxon, Mann, and Whitney U-test) as ear ly as 8 days after starting the 18 

### test. This effect was intensified in the presence of infection and additional thermo motor stress. 19



### No differences in bronchoalveolar cells were observ ed in pigs exposed to either 35 or 71 mg/m 3 20 

### compared to controls. However, a significantly hig her proportion of neutrophils was detected in 21 

### lavage fluid from infected pigs exposed to 35 mg/m 3 

### ammonia compared with infected controls 22 

### at 7 days postinfection ( α = 0.02, Wilcoxon, Mann, and Whitney U-test). Unde r additional 23 

### stress, the proportion of neutrophils at 7 days pos t infection was slightly less than in pigs that 24 

### were not put under additional stress, but remained significantly elevated above controls ( α = 25 

### 0.10, Wilcoxon, Mann, and Whitney U-test). In addi tion, the proportion of alveolar 26 

### macrophages in the lavage fluid of pigs exposed to 35 mg/m 3 

### ammonia was significantly lower 27 

### in infected pigs ( α = 0.05, Wilcoxon, Mann, and Whitney U-test) and in infected pigs subjected 28 

### to the swimming test ( α = 0.10, Wilcoxon, Mann, and Whitney U-test) at pos tinfection day 7. 29 

### Under these conditions, pigs exposed to 71 mg/m 3 

### demonstrated similar differentiation in 30 

### bronchoalveolar cells with controls at postinfectio n day 7. However, significant differences from 31 

### controls in the proportions of alveolar macrophages and neutrophils were observed at 32 

### postinfection day 3 among these pigs. In summary, results of the experiments conducted by 33 

### Neumann et al. (1987) suggested that ammonia exposu re in young pigs has the potential to 34 

### reduce systemic resistance to infection as evidence d by lower serum gamma globulin 35 

### concentrations and elevated nucleolar activity of p eripheral lymphocytes among exposed piglets. 36 

### Local resistance to infection in the lungs measured by pulmonary accumulation of aerosol 37 

### particles and quantitative cytology of the broncho- alveolar space was also reduced in piglets 38

### 46 DRAFT - DO NOT CITE OR QUOTE

exposed to ammonia. This study suggests that ammon ia toxicity may play an important role in 1 

### infectious respiratory diseases of swine. 2 

### F344/N rats (48/group, sex not specified) inoculate d with M. pulmonis were exposed to 3 

### ammonia concentrations of 0 or 100 ppm (0 or 71 mg/ m 3 

### ) for 3, 5, 7, or 9 days (Pinson et al., 4 

### 1986). Following the exposure periods, rats were k illed and their respiratory organs were 5 

### collected for histological examination. Ammonia-ex posed rats exhibited hyperplastic and 6 

### degenerative changes in the anterior nasal epitheli um. Lesions of mycoplasmosis were more 7 

### severe in ammonia-exposed rats than controls. 8 

### Gustin et al. (1994) injected endotoxin ( E. coli O127:B8) into the ventilated perfused 9 

### lungs collected from 7 to 23 male Belgian Landrace pigs exposed to ammonia concentrations of 10 

### 0, 25, 50, or 100 ppm (0, 18, 35, or 71 mg/m 3 

### ) ammonia for 6 days. Lethargy and decreased 11 

### body weight gain were observed at all ammonia conce ntrations. Blood samples taken on the first 12 

### and last day of the exposure period revealed no cha nges in the level of cortisol, total leukocyte 13 

### numbers, or in differential leukocyte percentages. No significant differences were observed on 14 

### the baseline pulmonary hemodynamics and microvascul ar permeability in perfused lungs from 15 

### any control or exposed pigs (see Section 4.4.2). H owever, the endotoxin-induced vascular 16 

### reaction was strongly altered in lungs from pigs ex posed to 100 ppm (71 mg/m 3 

### ) where the 17



### endotoxin-induced increase in total blood flow resi stance was completely abolished. The 18 

### vascular response to endotoxin was also reduced in pigs exposed to 50 ppm (35 mg/m 3 

### ). In 19 

### summary, concentrations of ammonia >50 ppm (35 mg/m 3 

### ) were shown to modulate the 20 

### pulmonary vascular response to endotoxins in pig lu ngs in vitro. 21

22



### 4.5. MECHANISTIC STUDIES 23 

### Studies have been conducted to investigate the mech anisms by which ammonia induces 24 

### irritation and effects on the gastric mucosa. A li mited number of studies have looked at the 25 

### potential for ammonia to induce genetic changes, al though the available studies of ammonia 26 

### genotoxicity are inadequate to characterize the gen otoxic potential of this compound. 27



### Studies conducted to examine the mechanisms of amm onia-related effects on the central 28 

### nervous system and kidney were largely conducted us ing ammonium salts (ATSDR, 2004); 29 

### however, as discussed in Section 4, because it is u nclear the extent to which the anion can 30 

### influence the toxicity of the ammonium salt, these mechanistic studies are not further reviewed 31



### in this assessment. A more detailed review of mech anistic studies of ammonia is provided as 32 

### supplemental information in Appendix C.6. 33

34



### 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 35



### 4.6.1. Oral 36 

### The only available data for oral exposure to ammoni a in humans consists of case reports 37 

### of individuals involved in the ingestion of househo ld cleaning solutions containing ammonia or 38

### 47 DRAFT - DO NOT CITE OR QUOTE

biting into capsules of ammonia smelling salts (Dwo rkin et al., 2004; Rosenbaum et al., 1998; 1 

### Christesen, 1995; Wason et al., 1990; Lopez et al., 1988; Klein et al., 1985; Klendshoj and 2 

### Rejent, 1966). Clinical signs reported in the case studies included headache, stomachache, 3 

### nausea, dizziness, diarrhea, drooling, erythematous and edematous lips, reddened and blistered 4 

### tongues, dysphagia, vomiting, oropharyngeal burns, laryngeal and epiglottal edema, erythmatous 5 

### esophagus with severe corrosive injury, and hemorrh agic esophago-gastro-duodeno-enteritis. 6 

### Oral toxicity data in animals are limited to 4- and 8-week drinking water studies that 7 

### examined the effects of ammonia on rat gastric muco sa (Tsujii et al., 1993; Kawano et al. 1991), 8 

### a gavage study in rabbits exposed to ammonium hydro xide for up to 142 days (Fazekas, 1939), 9 

### and a lifetime drinking water study in mice (Toth, 1972). Kawano et al. (1991) reported that 10 

### ammonia administered to Sprague Dawley rats at conc entrations of 0, 0.01 and 0.1% for two or 11 

### four weeks resulted in a statistically significant decrease in mean antral mucosal thickness; the 12 

### magnitude of the effect increased with dose and dur ation of exposure. In a follow-up study, 13 

### Tsujii et al. (1993) reported that treatment with 0 .01% ammonia for increased epithelial cell 14 

### migration in the mucosa of the rat stomach, and in particular in the antrum, leading to a decrease 15 

### in mucosal thickness and to mucosal atrophy. Fazek as (1939) reported initial decreases in body 16 

### weights and blood pressure, and elevated adrenal we ights in rabbits exposed to ammonium 17 

### hydroxide at 0.5 or 1% in solution for up to 142 da ys. Histological changes in the adrenal cortex 18 

### were also reported. Toth (1972) conducted the only chronic oral study of ammonia in 19 

### experimental animals, but did not evaluate noncance r endpoints. 20

21



### 4.6.2. Inhalation 22 

### Human and animal data indicate that the primary eff ects of ammonia inhalation are 23 

### irritation and burns and that the primary targets o f ammonia toxicity are the respiratory tract, 24 

### eyes, and skin (Kerstein et al., 2001; George et al ., 2000; Latenser and Lucktong, 2000; Morgan, 25 

### 1997; de la Hoz et al., 1996; Leduc et al., 1992; H olness et al., 1989; Millea et al., 1989; Weiser 26 

### and Mackenroth, 1989; Burns et al., 1985; Flury et al., 1983; Price et al., 1983; Close et al., 27 

### 1980; Hatton et al., 1979; Sobonya, 1977; Verberk, 1977; Taplin et al., 1976; Couturier et al., 28 

### 1971; Heifer, 1971; Slot, 1938). 29 

### Studies documenting inhalation exposure to ammonia in humans include numerous case 30 

### reports following acute exposures to high concentra tions of ammonia (i.e., accidental spill or 31 

### release), controlled exposure studies involving exp osing volunteers to ammonia vapors to 32 

### evaluate irritation effects and changes in pulmonar y function, studies in livestock farmers, and 33 

### studies of occupational exposures. Effects reporte d in case reports of human exposure included 34 

### acute point-of-contact irritation effects (e.g., bu rns to body surfaces, mouth, and respiratory tract) 35 

### and delayed effects including restricted pulmonary function (see Section 4.1.1 and Appendix 36 

### C.2). Inhalation effects in these studies were oft en accompanied by dermal and ocular irritation. 37 

### Primary effects reported in volunteer studies were eye, nose, and throat irritation (see Section 38

### 48 DRAFT - DO NOT CITE OR QUOTE

4.1.2 and Appendix C.3). Pulmonary function was no t affected in volunteers acutely exposed to 1 

### ammonia concentrations as high as 71 mg/m 3 

### (100 ppm). Studies comparing the pulmonary 2 

### function of asthmatics and healthy volunteers expos ed to ammonia do not suggest that asthmatics 3 

### are more sensitive to the pulmonary effects of ammo nia. Studies in livestock farmers 4 

### demonstrated an association between ammonia exposur e and impaired respiratory function, but 5 

### ammonia exposures in these studies were generally c onfounded by concomitant exposures to 6 

### airborne dust, bacteria, fungal spores, endotoxin, and mold (see Section 4.1.3 and Appendix 7 

### C.4). Studies of industrial exposures in workers r eported pulmonary effects including coughing, 8 

### wheezing, phlegm, chest tightness, shortness of bre ath, and decreased respiratory function (Table 9 

### 4-12) (Rahman et al., 2007; Ali, 2001; Ballal et al ., 1998; Holness et al., 1989). 10



### There are no chronic inhalation studies of ammonia in animals. The observed effects in 11



### subchronic animal studies in which ammonia was admi nistered via inhalation included 12 

### inflammatory changes in the nasal mucosa and trache al epithelium of rats and pigs (Broderson et 13 

### al., 1976; Doig and Willoughby, 1971), and histolog ical changes in the livers, kidneys, lung, 14 

### spleen, and adrenal gland of rats and guinea pigs ( Coon et al., 1970; Weatherby, 1952) 15 

### (Table 4-12). Exposure durations in these studies ranged from 5 to 18 weeks. NOAELs and 16 

### LOAELs did not vary greatly across species and stra ins. LOAELs ranged from 18 to 127 mg/m 3 17 

### and NOAELs ranged from 7 to 53 mg/m 3 

### . In three studies, effects were observed at the l owest 18 

### dose, and NOAELs could not be identified. In the o nly available study of developmental toxicity 19 

### of ammonia, no maternal or developmental effects we re observed in pigs following gestational 20 

### exposure to ammonia concentrations up to 25 mg/m 3 

### (Diekman et al., 1993). 21 

### Acute and short-term studies in animals following i nhalation exposure demonstrate eye 22 

### and nose irritation, dyspnea, ataxia, seizures, com a, and death in rats and mice (rat LC 50 

### values 23



### ≥ 11,590 mg/m 3 

### ; mouse LC 50 

### values ≥ 2,990 mg/m 3 

### ), decreased pulmonary function with 24 

### decreased respiratory rate in rats at concentration s ≥ 848 mg/m 3 

### , mice at concentrations 25



### ≥ 214 mg/m 3 

### , and in rabbits at concentrations ≥ 35 mg/m 3 

### . Histopathological changes in the 26 

### respiratory tract following acute exposure to ammon ia include irritation and inflammation of the 27 

### nasal and tracheal epithelium, pneumonitis, atelect asis, and intralveolar hemorrhage of the lower 28 

### lung. 2949 DRAFT - DO NOT CITE OR QUOTETable 4-12. Summary of noncancer results of humanoccupational studies and repeat-dose studies in experimentalanimals involving inhalation exposure to ammoniaExposedpopulationExposureconcentration(mg/m3)/ durationNOAEL(mg/m3)LOAEL(mg/m3)Effects at theLOAELconcentration Comment ReferenceOccupational worker studiesIndustrialworkers (58 maleworkers and 31male controlsfrom office area)low (<4.4),medium(4.4–8.8), and high(>8.8)avg exposure: 12 y8.8(adjusted: 3.1)aNot determinedCross-sectional study in soda ash plant. Nodifferences in pulmonary function or subjectivesymptomology relative to the control groupHolness et al.,1989Industrialworkers (63ammonia plantand 77 urea plantworkers; 25controls fromadministrationbuilding)ammonia plant(mean): 4.9urea plant (mean):18.5mean employmentduration: 16 y4.9 18.5 Increased prevalenceof respiratorysymptoms anddecrease in lungfunctionCross-sectional study in urea fertilizer factory.Concentrations in this table were based on the PACIII method; concentrations measured using theDräger tube method were 4-5 times higher.Rahman et al.,2007Experimental animal studiesRat (SpragueDawley andLongs-Evans)(15/sex/group)0, 40, 127, 262,455, or 470 for 90–114 d40 127 Nonspecificinflammatorychanges in the lungsand kidneysNasal discharge and nonspecific circulatory anddegenerative changes in the lungs and kidneys wereobserved at 262 mg/m3(not further described,incidence not reported). Frank-effect-level of 455mg/m3based on 90–98% mortalityCoon et al., 1970Sherman rat(5/sex/group)7 or 106 for 75 d 7 106 Nasal lesionsBroderson et al.,1976F344 rat(6/sex/group)0, 18, 35, 71, or 177for up to 49 dNot determined 18 Increased severity ofrhinitis, otitis media,tracheitis, andpneumonia, andincreased incidenceof respiratory lesionsRats were exposed continuously to ammonia for 7days prior to inoculation withM. pulmonisand 28–42 days following inoculationBroderson et al.,1976Guinea pigs(12 guinea pigs,sex notspecified)0 or 120, 6 hrs/d,5 d/wk for 18 wksNot determined 120 Congestion of thespleen, liver, andkidneys and earlydegenerative changesin the adrenal glandWeatherby, 195250 DRAFT - DO NOT CITE OR QUOTETable 4-12. Summary of noncancer results of humanoccupational studies and repeat-dose studies in experimentalanimals involving inhalation exposure to ammoniaExposedpopulationExposureconcentration(mg/m3)/ durationNOAEL(mg/m3)LOAEL(mg/m3)Effects at theLOAELconcentration Comment ReferenceYorkshireLandrace pig(6/group, sex notspecified)0 or 71 for 6 wks Not determined 71 Thickening of thetracheal epitheliumwith a concomitantdecrease in thenumber of trachealepithelial goblet cellsThe mean concentration of ammonia in the controlchamber was measured at 5.6 mg/m3Doig andWilloughby, 1971Pig(4–8/group, sexnot specified)0, 35, or 53 for 109d53 Not identifiedCurtis et al., 1975Pig(24 pigs/group,several breeds,sex not reported)0, 0.4, 7, 13.3, or 26for 5 wks26 Not identified Exposures were to ammonia at concentrations of 0,0.4, 7, 13.3, or 26 mg/m3and to inhalable dust at1.2,2.7, 5.1, or 9.9 mg/m3; all pigs including controlsdemonstrated infections common to pigs oncommercial farmsDone et al., 2005aAdjusted to continuous exposure as follows: NOAELADJ= NOAEL × VEho/VEh× 5 days/7 days = 8.8 mg/m3× 10 m3/20 m3× 5 days/7 days = 3.1 mg/m3, where:VEho= human occupational default minute volume and VEh= human ambient default minute volume.

### 51 DRAFT - DO NOT CITE OR QUOTE

4.6.3. Mode-of-Action Information 1 

### Inhalation exposure to ammonia is associated with u pper respiratory irritation in humans 2 

### and animals. Ammonia interacts with moisture along the respiratory tract to form ammonium 3 

### hydroxide, which causes necrosis of tissue through saponification leading to inflammation 4 

### (Amshel et al., 2000; Jarudi and Golden, 1973). 5



### Oral studies with H. pylori suggest possible mechanisms by which ammonia may c ause 6 

### gastric mucosal damage, namely, increased cell vacu olation and decreased viability of the cells 7 

### (Mégraud et al., 1992), increased release of endoth elin-1 and thyrotropin releasing hormone from 8 

### the gastric mucosa (Mori et al., 1998), release of cysteine proteases in the stomach that 9 

### contribute to the development of gastric hemorrhagi c mucosal lesions (Nagy et al., 1996) and 10 

### apoptosis directed by mitochondrial membrane destru ction (Suzuki et al., 2000). 11

12



### 4.7. EVALUATION OF CARCINOGENICITY 13 

### 4.7.1. Summary of Overall Weight-of-Evidence 14



### Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is 15 

### “inadequate information to assess the carcinogenic potential” of ammonia based on the absence 16 

### of ammonia carcinogenicity studies in humans, and a single lifetime drinking water study of 17 

### ammonia in mice that showed no evidence of carcinog enic potential. 18

19 

### 4.7.2. Synthesis of Human, Animal, and Other Suppo rting Evidence 20 

### Human data on the carcinogenic effects of ammonia a re not available. Animal 21 

### carcinogenicity data are limited. Toth (1972) did not observe any evidence for carcinogenicity 22 

### of ammonium hydroxide administered orally to mice o ver a lifetime. Tsujii et al. (1995, 1992) 23 

### suggest that ammonia administered in drinking water may act as a cancer promoter in H. pylori 24 

### induced gastric cancer. In these studies (Tsujii e t al., 1995, 1992), rats dosed with ammonia and 25 

### pretreated with MNNG had a greater incidence of gas tric cancer and number of tumors per 26 

### tumor-bearing rat than rats receiving only MNNG and tap water (Tsujii et al., 1995, 1992), 27 

### suggesting that ammonia can act as a tumor promoter . The available studies of ammonia 28 

### genotoxicity are inadequate to characterize the gen otoxic potential of this compound. 29

30



### 4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 31 

### 4.8.1. Possible Childhood Susceptibility 32 

### There are no studies of the toxicity of ammonia in children comparing them to any other 33 

### life stages that would support an evaluation of chi ldhood susceptibility. Case reports of acute 34 

### exposure of children (<18 years old) to ammonia are summarized in Table C-1 (Dilli et al., 2005; 35 

### Dworkin et al., 2004; Rosenbaum et al., 1998; Wason et al., 1990; Millea et al., 1989; Lopez et 36 

### al., 1988; Klein et al., 1985; Close et al., 1980; Hatton et al., 1979; Helmers et al., 1971; Levy et 37 

### al., 1964); however, these studies do not provide i nformation useful for evaluation of 38

### 52 DRAFT - DO NOT CITE OR QUOTE

susceptibility from chronic low-level exposure to a mmonia. No experimental animal studies of 1 

### ammonia were identified that compared effects in ju venile animals to adults that could be used to 2 

### inform childhood susceptibility in humans. 3

4



### 4.8.2. Possible Gender Differences 5 

### There are no studies of gender differences in susce ptibility to the toxic effects of 6 

### ammonia; information from the available toxicity st udies of ammonia provides no evidence of 7 

### gender differences. 8

9 

### 4.8.3. Other Susceptible Populations 10 

### Persons who suffer from severe liver or kidney dise ase (including cirrhosis, acute renal 11 

### failure, or liver failure) may be more susceptible to ammonia intoxication (hyperammonemia), as 12 

### it is chiefly by the actions of these organs that a mmonia is biotransformed and excreted (Córdoba 13 

### et al., 1998; Gilbert, 1988; Jeffers et al., 1988; Souba, 1987). Individuals with hereditary urea 14 

### cycle disorders are also at risk (Schubiger et al., 1991). In these disease conditions, it is the bra in 15 

### where ammonia is most toxic, and the elevated ammon ia levels that accompany human diseases 16 

### such as acute liver or renal failure can predispose an individual to encephalopathy; these effects 17 

### are especially marked in newborn infants (Miñana et al., 1995; Souba, 1987). 18 

### Individuals with respiratory disease (e.g., asthma) also could represent a susceptible 19 

### population; however, controlled human studies that examined both healthy volunteers and 20 

### volunteers with asthma, as well as cross sectional studies of livestock farmers, exposed to 21 

### ammonia (Petrova et al., 2008; Sigurdarson et al., 2004; Vogelzang et al., 2000, 1998, 1997; 22 

### Preller et al., 1995) generally did not observe a g reater sensitivity to respiratory effects in 23 

### populations with underlying respiratory disease. H owever, the findings from an epidemiological 24 

### study of a group of workers chronically exposed to airborne ammonia indicated that ammonia 25 

### inhalation can exacerbate existing symptoms, includ ing cough, wheeze, nasal complaints, eye 26 

### irritation, throat discomfort, and skin irritation (Holness et al., 1989). 27

### 53 DRAFT - DO NOT CITE OR QUOTE 5. DOSE-RESPONSE ASSESSMENTS 1

2

3 

### 5.1. ORAL REFERENCE DOSE (RfD) 4



### An RfD for ammonia was not derived because the avai lable oral data for ammonia were 5 

### considered insufficient to support derivation of a chronic reference value. As discussed in 6 

### Sections 4.1.1, 4.2.1, and 4.6.1, oral toxicity dat a in humans are limited to case reports of 7 

### individuals suffering from acute neurological (e.g. , headache, dizziness) and gastrointestinal 8 

### (e.g., stomach ache, nausea, diarrhea, distress and burns along the digestive tract) effects from 9 

### ingesting household cleaning solutions containing a mmonia or biting into capsules of ammonia 10 

### smelling salts. In animals, the only chronic toxic ity study of ammonia is the lifetime 11 

### carcinogenicity drinking water study in mice by Tot h (1972) that reported tumor incidence only 12 

### and did not provide noncancer data to support devel opment of an RfD. A subchronic gavage 13 

### study in rabbits exposed to ammonium hydroxide for up to 142 days (Fazekas, 1939) is available 14 

### but was inadequate for deriving an oral RfD because of limited reporting of study details and 15 

### results, as well as inadequate study design. The o nly remaining oral study is an eight-week 16 

### drinking water study in Sprague Dawley rats (Tsujii et al., 1993) that examined the effects of 17 

### ammonia on the gastric mucosa. 18 

### Tsujii et al. (1993) found that ammonia at a concen tration of 0.01% (equivalent to a daily 19 

### dose of 33 mg/kg-day) increased epithelial cell mig ration in the mucosa of the stomach (in 20 

### particular the antrum) leading to a statistically s ignificant decrease in the thickness of the antral 21 

### mucosa at 4 and 8 weeks of treatment; there was no effect on the body mucosa. The authors 22 

### reported that the gastric mucosal effects observed in rats resemble mucosal changes in human 23 

### atrophic gastritis. EPA identified a LOAEL for the Tsujii et al. (1993) study of 33 mg/kg-day 24 

### based on decreased gastric mucosal thickness, an ef fect considered by EPA to be adverse; a 25 

### NOAEL was not identified. The Tsujii et al. (1993) study and decreased antral mucosal 26 

### thickness were considered as a potential principal study and critical effect. EPA identified a 27 

### potential point of departure (POD) based on the LOA EL of 33 mg/kg-day; BMD modeling was 28 

### not utilized because this single-concentration stud y is not amenable to dose-response analysis. 29 

### In EPA’s guidance document entitled, Recommended Use of Body Weight 3/4 

### as the 30 

### Default Method in Derivation of the Oral Reference Dose (U.S. EPA, 2011 a 

### ), the Agency 31 

### endorses a hierarchy of approaches for converting d oses administered orally to laboratory animal 32 

### species to human equivalent oral exposures in deriv ing the RfD, with the preferred approach 33 

### being physiologically-based toxicokinetic modeling. An alternate approach includes using 34 

### chemical-specific information in the absence of a c omplete physiologically-based toxicokinetic 35



### model. In lieu of a toxicokinetic model or chemica l-specific data to inform the generation of 36 

### human equivalent oral exposures, EPA endorses body weight scaling to the 3⁄4 power (i.e., BW 3/4 

### ) 37 

### as a default to extrapolate toxicologically equival ent doses of orally administered agents from 38

### 54 DRAFT - DO NOT CITE OR QUOTE

laboratory animals to humans for the purpose of der iving an RfD. When BW 3/4 

### scaling is used in 1 

### deriving the RfD, EPA also advocates a reduction in the interspecies uncertainty factor (UF A 

### ) 2 

### from 10 to 3, as BW 3/4 

### scaling addresses predominantly toxicokinetic (and some toxicodynamic) 3 

### aspects of the UF A



### . 4 

### The guidance raises some important uncertainties in applying allometric (more 5 

### specifically BW 3/4 

### ) scaling when the critical effect used to derive t he reference value is a portal6 

### of-entry effect. No physiologically-based pharmaco kinetic model or chemical-specific 7 

### information exists to inform the generation of huma n equivalent oral exposures for ammonia. 8 

### Furthermore, the potential critical effect (i.e., d ecreased gastric antral mucosal thickness) is 9 

### considered a portal-of-entry effect based on the fo llowing. Tsujii et al. (1993) postulated that the 10 

### difference in response of the mucosa in the stomach body versus the mucosa of the antrum 11 

### relates to differences in pH in the two stomach reg ions. Most ammonia is transformed to 12 

### ammonium ion in solution at physiological pH; the r atio of ammonia to ammonium ion increases 13 

### 10-fold with each unit rise in pH. In the mucosa o f the stomach body—an acid-secreting 14 

### mucosa—ammonia is protonated to the ammonium ion, w hich reduces the cytotoxicity 15



### associated with nonionized ammonia. In the antral mucosa—a nonacid secreting area of the 16 

### stomach—pH is higher, resulting in a relatively hig her concentration of ammonia and thus 17 

### enhanced cytotoxicity. Because ammonia toxicity ap pears to be a function of the 18 

### physical/chemical environment at the mucosal surfac e (i.e., a portal-of-entry effect) and it is not 19 

### clear if regions of the stomach scale allometically across species, a surface area adjustment 20 

### would be the most relevant for interspecies extrapo lation; however, a dose scaling approach 21 

### involving mass per unit surface area has not been d eveloped (U.S. EPA, 2011a). Therefore, 22 

### because effects on the gastric antral mucosa are no t expected to scale allometrically, a 23



### BW 3/4 

### scaling approach has not been applied as a default approach (in combination with a 24 

### reduced default UF for interspecies extrapolation). 25 

### An RfD is derived by dividing the POD by a composit e uncertainty factor (UF). The 26 

### UFs, selected based on EPA’s A Review of the Reference Dose and Reference Concen tration 27



### Processes (U.S. EPA, 2002; Section 4.4.5), address five area s of uncertainty. Considering the 28 

### available oral data, the composite UF for ammonia w ould be 10,000. In the report, A Review of 29 

### the Reference Dose and Reference Concentration Proc esses (U.S. EPA, 2002), the RfD/RfC 30 

### technical panel concluded that, in cases where maxi mum uncertainty exists in four or more areas 31 

### of uncertainty, or when the total uncertainty facto r is 10,000 or more, it is unlikely that the 32



### database is sufficient to derive a reference value. Therefore, consistent with the 33 

### recommendations in U.S. EPA (2002), the available o ral data for ammonia were considered 34 

### insufficient to support reference value derivation and an RfD for ammonia was not derived. 35 

### Route-to-route extrapolation of the inhalation data was considered for deriving the oral 36 

### RfD; however, in the absence of a PBPK model and be cause the critical effect from the 37

### 55 DRAFT - DO NOT CITE OR QUOTE

inhalation literature is a portal-of-entry effect ( respiratory symptoms and lung function changes), 1 

### route-to-route extrapolation is not supported. 2

3 

### 5.1.1. Previous RfD Assessment 4 

### No RfD was derived in the previous IRIS assessment for ammonia. 5

6 

### 5.2. INHALATION REFERENCE CONCENTRATION (RfC) 7 

### 5.2.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification 8 

### Inhalation studies of ammonia exposure in humans in clude numerous case reports 9 

### following acute exposures to high concentrations (e .g., accidental spills/releases), controlled 10 

### exposure studies involving volunteers exposed to am monia vapors for short periods of time to 11 

### evaluate irritation effects and changes in pulmonar y function, studies in livestock farmers, and 12 

### studies of industrial occupational exposures compar ing the prevalence of acute respiratory 13 

### symptoms and changes in lung function between expos ed and nonexposed worker populations. 14 

### Studies in experimental animals (including rats, gu inea pigs, and pigs) have also examined 15 

### respiratory and other systemic effects of ammonia f ollowing subchronic inhalation exposures. 16 

### Acute exposure studies (i.e., case reports, control led volunteer studies) involved 17 

### exposures too brief in duration to be used for deri vation of a chronic RfC. Further, case reports 18 

### of acute exposure do not typically have the appropr iate exposure information necessary for a 19 

### valid evaluation of dose response. Studies of live stock farmers are also not suitable for dose20 

### response assessment because of multiple and possibl y confounding exposures to dusts, 21 

### endotoxins, bacteria, fungi, molds, and other chemi cals (including potentially volatile chemicals 22 

### for which monitoring data have not been collected). 23 

### There are two occupational studies of ammonia expos ure in workers for which 24 

### quantitative dose-response information is available . Holness et al. (1989) reported similar odor 25 

### detection thresholds, lung function, and prevalence in the reporting of acute respiratory 26 

### symptoms between workers exposed to a mean TWA ammo nia concentration of 9.2 ppm 27 

### (6.5 mg/m 3 

### ) and a nonexposed worker population. When the exp osed workers were grouped into 28 

### three exposure categories (high: ≥ 12.5 ppm [8.8 mg/m 3 

### ], medium: 6.25–12.5 ppm [4.4– 29 

### 8.8 mg/m 3 

### ], and low: ≤ 6.25 ppm [4.4 mg/m 3 

### ]), no statistically significant differences ( p < 0.05) 30 

### in symptoms or changes in lung function between the groups were evident. Based on lack of 31 

### symptomology and changes in lung function, EPA iden tified a NOAEL of 12.5 ppm (8.8 mg/m 3 

### ) 32 

### for the Holness et al. (1989) study based on the gr ouping of workers into three exposure 33 

### categories. In a more recent occupational study, R ahman et al. (2007) reported a significantly 34 

### higher prevalence of acute respiratory symptoms amo ng workers exposed to 26.1 ppm 35 

### (18.5 mg/m 3 

### ) ammonia in the urea plant of a fertilizer factory than in workers exposed to 36 

### 6.9 ppm (4.9 mg/m 3 

### ) ammonia in the ammonia plant or nonexposed worker s in the 37 

### administration building. Furthermore, workers in t he urea plant demonstrated significant cross- 38

### 56 DRAFT - DO NOT CITE OR QUOTE

shift (comparing preshift measurements to postshift measurements) changes in lung function 1 

### (i.e., FVC and FEV decreased significantly). A sim ilar cross-shift change in lung function was 2 

### not observed among workers in the ammonia plant. E PA identified a NOAEL (4.9 mg/m 3 

### ) and a 3 

### LOAEL (18.5 mg/m 3 

### ), based on increased prevalence of respiratory sym ptoms and decrease in 4 

### lung function from the Rahman et al. (2007) study. The findings from Holness et al. (1989) and 5 

### Rahman et al. (2007) are supported by the results f rom the cross-sectional studies of fertilizer 6 

### factory workers by Ballal et al. (1998) and Ali (20 01), which suggest that occupational exposure 7 

### to ammonia concentrations >18 mg/m 3 

### are associated with respiratory symptoms and alter ed 8 

### pulmonary function. 9 

### Figure 5-1 compares effect levels from studies of w orkers occupationally exposed to 10



### ammonia with those from studies of human volunteers acutely exposed to ammonia. As shown 11 

### in this figure, irritation effects in controlled hu man volunteer studies were generally observed at 12 

### concentrations higher than those that induced respi ratory symptoms in industrially exposed 13 

### workers; however, Sundblad et al. (2004) reported e ye irritation and other symptoms at a 14 

### concentration below effect levels reported in occup ationally-exposed populations. Effects in the 15 

### Sundblad et al. study were transient, and the inves tigators noted a tendency towards adaptation. 16 

### Nevertheless, it is interesting to note that effect levels in acute inhalation studies may occur in 17 

### the same concentration range as those in occupation al exposure settings. 1857 DRAFT - DO NOT CITE OR QUOTE1Figure 5-1. Exposure-response array comparing workers occupationally exposed and human volunteers acutely2exposed to ammonia.30.010.11101001000PrevalenceofrespiratorysymptomsandlungfunctionHolnesset al.(1989)PrevalenceofrespiratorysymptomsanddecreasedlungfunctionRahmanet al.(2007)Cough,lacrimation,nose andthroatirritationSilvermanet al.(1949)EyeirritationMacEwenet al.(1970)No effecton lungfunctionColeet al.(1977)Lacrimation,transientdiscomfortFergusonet al.(1977)Eyeirritation,odordetection,headache,dizziness,feeling ofintoxicationSundbladet al.(2004)EyeirritationVerberk(1977)NasalirritationPetrovaet al.(2008)EyeirritationPetrovaet al.(2008)Severe eye,nose andthroatirritationVerberk(1977)LungfunctionVerberk(1977)LungfunctionSigurdarsonet al.,(2004)LungfunctionSundbladet al.,(2004)BronchoconstrictionDouglasand Coe(1987)Habituationto acuteirritationandrespiratorysymptomsIhrig et al.(2006)Odordetection,moderate tostrongirritationAltmannet al.(2006)INDUSTRIAL STUDIESCONTROLLED ACUTE EXPOSURE STUDIES

Exposure (mg/m 3 )NOAELLOAELVertical lines show range ofexposures in study. Closedcircles show exposures tested instudy.

### 58 DRAFT - DO NOT CITE OR QUOTE

Animal studies of inhalation exposure to anhydrous ammonia include subchronic studies 1 

### that reported inflammatory changes in nasal mucosa and tracheal epithelium (Gaafar et al., 1992; 2 

### Broderson et al., 1976; Doig and Willoughby, 1971), and histological changes in liver and 3 

### kidneys (Coon et al., 1970; Weatherby, 1952). NOAE L and LOAEL values could not be 4



### identified for a number of these studies. Studies for which effect levels could be derived are 5 

### summarized in Table 4-12. Figure 5-2 is an exposur e-response array comparing inhalation 6 

### exposure to ammonia in humans (occupational exposur e studies) and animals. As shown, effects 7



### in animals are observed near or above the LOAEL obs erved in the human studies. 859 DRAFT - DO NOT CITE OR QUOTE1Figure 5-2. Exposure-response array comparing noncancer effects in occupationally exposed workers and2experimental animals exposed to ammonia by inhalation.30.010.11101001000Prevalence ofrespiratory symptoms& lung function (maleoccupational);Holness et al. (1989)Prevalence ofrespiratory symptoms& acute decreasedlung function(occupational);Rahman et al. (2007)Upper respiratorytract - structuraldamage (pig); Doigand Willoughby(1971)Nasal lesions (rat);Broderson et al.(1976)Respiratory lesions(rat); Broderson et al.(1976)Respiratory infectionin presence ofinhalable dust (pig);Done et al. (2005)Nonspecificinflammatorychanges in lungs andkidneys (rat); Coon etal. (1970)Congestion of spleen,liver & kidneys, earlydegenerativechanges in adrenalgland (guinea pig);Weatherby (1952)Weight gain &histological lesions ofrespiratory tract &visceral organs(pig); Curtis et al.(1975)HUMAN STUDIESEXPERIMENTAL ANIMAL STUDIES

Exposure (mg/m 3 )LOAELNOAELFELIntermediate doseVertical lines showrange of exposuresin study. Closedcircles showexposures tested in

### 60 DRAFT - DO NOT CITE OR QUOTE

As adequate data on the effects of inhalation expos ure to ammonia are available in 1 

### humans for deriving an RfC, animal data are conside red in this assessment as supportive. Use of 2



### human data to derive the RfC also avoids the uncert ainty associated with interspecies 3 

### extrapolation introduced when animal data are used as the basis for the RfC. The two 4 

### occupational exposure studies of ammonia by Holness et al. (1989) and Rahman et al. (2007) 5 

### which examined respiratory symptoms and effects on lung function provide consistent estimates 6 

### of the effect level for ammonia, with the NOAEL of 8.8 mg/m 3 

### identified from the Holness et al. 7 

### (1989) study falling between the NOAEL and LOAEL va lues (4.9 mg/m 3 

### and 18.5 mg/m 3



### , 8 

### respectively) from the Rahman et al. (2007) study. The Holness et al. (1989) study was selected 9 

### as the principal study for deriving an RfC over the Rahman et al. (2007) study because it 10 

### identified the higher NOAEL of these two occupation al studies. Consideration of analytical 11 

### methods also supports the selection of Holness et a l. (1989) as the principal study. Rahman et al. 12 

### (2007) used two analytical methods for measuring am monia concentrations in workplace air 13 

### (Dräger PAC III and Dräger tube); concentrations me asured by the two methods differed by 14 

### four- to five-fold, indicating some uncertainty in these measurements. In contrast, the Holness et 15 

### al. (1989) study used an established analytical met hod of measuring exposure to ammonia 16 

### recommended by NIOSH that involved the collection o f air samples on ATSG absorption tubes. 17 

### Therefore, the Holness et al. (1989) study was sele cted as the principal study, and 18 

### increased respiratory symptoms and decreased lung f unction, considered by EPA to be adverse, 19



### were selected as the critical effect. 20

21 

### 5.2.2. Methods of Analysis 22 

### EPA identified a NOAEL of 8.8 mg/m 3 

### , based on the lack of increased respiratory 23 

### symptoms and decreased lung function in Holness et al. (1989) and selected this as the point of 24 

### departure (POD) for RfC derivation. This NOAEL was identified as the POD in the context of 25 

### the entire ammonia database, including other occupa tional studies that reported pulmonary 26 

### effects at higher workplace ammonia concentrations (i.e., the LOAEL of 18.5 mg/m 3 

### identified 27 

### by Rahman et al. (2007)). Additionally, effects in animals are observed near or above the 28 

### LOAEL observed in human studies, thereby supporting the occupational epidemiology studies. 29

30 

### 5.2.3. RfC Derivation—Including Application of Unc ertainty Factors (UFs) 31 

### The NOAEL of 8.8 mg/m 3 

### identified in the Holness et al. (2007) study is us ed as the POD 32



### for RfC derivation. Since this POD is based on occ upational exposure, the value was first 33 

### adjusted for continuous exposure as follows: 34

35



### NOAEL ADJ 

### = NOAEL × VE ho 

### /VE h 

### × 5 days/7 days 36 

### = 8.8 mg/m 3 

### × 10 m 3 

### /20 m 3 

### × 5 days/7 days 37 

### = 3.1 mg/m 3 38

### 61 DRAFT - DO NOT CITE OR QUOTE

Where: 1



### VE ho 

### = human occupational default minute volume (10 m 3 

### breathed during the 8-hour 2 

### workday, corresponding to a light to moderate activ ity level [U.S. EPA, 2011b]) 3



### VE h 

### = human ambient default minute volume (20 m 3 

### breathed during the entire day) 4

5 

### The UFs, selected based on EPA’s A Review of the Reference Dose and Reference 6



### Concentration Processes (U.S. EPA, 2002; Section 4.4.5), address five areas of uncertainty 7 

### resulting in a composite UF of 10. This composite uncertainty factor was applied to the selected 8



### POD to derive an RfC. 9



### • An intraspecies uncertainty factor, UF H 

### , of 10 was applied to account for potentially 10 

### susceptible individuals in the absence of data eval uating variability of response to 11 

### inhaled ammonia in the human population. 12



### • An interspecies uncertainty factor, UF A 

### , of 1 was applied to account for uncertainty in 13 

### extrapolating from laboratory animals to humans bec ause the POD was based on 14 

### human data from an occupational study. 15



### • A subchronic to chronic uncertainty factor, UF S 

### , of 1 was applied because the 16 

### occupational exposure period in the principal study (Holness et al., 1989), i.e., mean 17 

### years at present job for exposed workers, of approx imately 12 years was of chronic 18



### duration. 19



### • A LOAEL to NOAEL uncertainty factor, UF L 

### , of 1 was applied because a NOAEL 20



### value was used as the POD. 21



### • A database uncertainty factor, UF D 

### , of 1 was applied to account for deficiencies in t he 22



### database. The ammonia inhalation database consists of a large number of case reports 23 

### of acute exposure to high ammonia concentrations (e .g., accidental spills/releases), 24 

### controlled exposure studies involving volunteers ex posed to ammonia vapors for 25 

### short periods of time to evaluate irritation effect s and changes in pulmonary function, 26 

### studies in livestock farmers, and studies of occupa tional exposure focused on effects 27 

### of ammonia on respiratory symptoms and lung functio n. Studies of the toxicity of 28 

### inhaled ammonia in experimental animals include sub chronic studies in rats, guinea 29 

### pigs, and pigs that examined respiratory and other systemic effects of ammonia and 30 

### one limited, reproductive toxicity study in young f emale gilts pigs. The database 31 

### lacks developmental and multigeneration reproductiv e toxicity studies. 32 

### Ammonia is endogenously produced in humans and ani mals during fetal and 33



### adult life and concentrations in blood are homeosta tically regulated to remain at low 34 

### levels. Baseline blood levels in healthy individua ls range from 0.1-1.0 μg/mL 35 

### (Monsen, 1987; Conn, 1972; Brown et al., 1957). Ev idence in animals (Manninen et 36 

### al., 1988; Schaerdel et al., 1983) suggests that ex posure to ammonia at concentrations 37 

### up to 18 mg/m 3 

### does not alter blood ammonia levels (see Section 3 ). Therefore, 38

### 62 DRAFT - DO NOT CITE OR QUOTE exposure at the POD (3.1 mg/m 3 

### ) associated with respiratory effects following 1 

### inhalation exposure would not be expected to alter ammonia homeostasis or result in 2



### measureable increases in blood ammonia concentratio ns. Thus, the concentration of 3 

### ammonia at the POD for the RfC would not be expecte d to result in systemic toxicity, 4 

### including reproductive or developmental toxicity. The fact that the fetoplacental unit 5 

### produces ammonia and that concentrations in human u mbilical vein and artery blood 6 

### (at term) have been shown to be higher than concent rations in maternal blood (see 7 

### Section 3) also provides assurance that development al toxicity would not be 8



### associated with concentrations of ammonia at or bel ow the POD. As noted in EPA’s 9 

### A Review of the Reference Dose and Reference Concen tration Processes (U.S. EPA, 10 

### 2002), “the size of the database factor to be appli ed will depend on other information 11 

### in the database and on how much impact the missing data may have on determining 12 

### the toxicity of a chemical and, consequently, the P OD.” Because the lack of two13 

### generation reproductive and developmental toxicity studies in the ammonia toxicity 14 

### database should not impact the determination of amm onia toxicity at the POD, a 15



### database UF to account for the lack of these studie s was not considered necessary. 16

17



### The RfC for ammonia was calculated as follows: 18

19 

### RfC = NOAEL ADJ 

### ÷ UF 20 

### = 3.1 mg/m 3 

### ÷ 10 21 

### = 0.3 mg/m 3 22

23



### 5.2.4. Previous RfC Assessment 24 

### The previous IRIS assessment for ammonia (posted to the database in 1991) presented an 25 

### RfC of 0.1 mg/m 3 

### based on co-critical studies—the occupational expo sure study of workers in a 26 

### soda ash plant by Holness et al. (1989) and the sub chronic study by Broderson et al. (1976) that 27 

### examined the effects of ammonia exposure in F344 ra ts inoculated on day 7 of the study with M. 28 

### pulmonis . The NOAEL of 6.4 mg/m 3 

### (mean concentration of the entire exposed group) f rom the 29 

### Holness et al. (1989) study (duration adjusted: NOA EL ADJ 

### = 2.3 mg/m 3 

### ) was used to derive the 30 

### RfC by applying a composite UF of 30, 10 to account for the protection of sensitive individuals 31



### and 3 for database deficiencies to account for the lack of chronic data, the proximity of the 32 

### LOAEL from the subchronic inhalation study in the r at (Broderson et al., 1976) to the NOAEL, 33 

### and the lack of reproductive and developmental toxi cology studies. A database UF larger than 3 34 

### was not applied because studies in rats (Schaerdel et al., 1983) that showed no increase in blood 35 

### ammonia levels at an inhalation exposure to 32 ppm and only minimal increases at 300–1000 36 

### ppm suggested that no significant distribution is l ikely to occur at the human equivalent 37



### concentration. 38

39

### 63 DRAFT - DO NOT CITE OR QUOTE

5.3. CANCER ASSESSMENT 1 

### As discussed in Section 4.7, data are “inadequate f or assessing the carcinogenic 2 

### potential” of ammonia, so no quantitative cancer as sessment was conducted. Also, no 3 

### carcinogenicity assessment was presented in the pre vious IRIS assessment. 4

### 64 DRAFT - DO NOT CITE OR QUOTE

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HA ZARD AND DOSE 1



### RESPONSE 2

3

4



### 6.1. HUMAN HAZARD POTENTIAL 5 

### Ammonia is a colorless gas that occurs naturally i n the environment. Ammonia is found 6 

### in water, soil, and air, and is a source of nitroge n for plants and animals. Most of the ammonia in 7



### the environment comes from the natural breakdown of animal waste and dead plants and 8 

### animals. Ammonia may also be present in the enviro nment as the result of its use as a fertilizer, 9 

### chemical intermediate, alkalizer, metal treating/ex traction agent, water treatment chemical or as 10 

### emissions from urea selective catalytic reduction a ftertreatment systems in diesel vehicles. If 11 

### compressed or in an aqueous solution, ammonia can o ccur as a liquid. Ammonia has a very 12 

### sharp odor that is familiar to most people because of its use in smelling salts and household 13 

### cleaners. In water, most of the ammonia is convert ed to the ionic form, ammonium ion. 14 

### Ammonium ions are not gaseous and have no odor. 15 

### Ammonia can be absorbed by both the inhalation and oral routes of exposure, but there is 16 

### less certainty regarding absorption through the ski n. Absorption through the eye has been 17



### documented. Most of the inhaled ammonia is retaine d in the upper respiratory tract and is 18 

### subsequently eliminated in expired air. Both exoge nous and endogenously produced ammonia 19



### are absorbed in the intestine. Ammonia that reache s the circulation is widely distributed to all 20 

### body compartments, although substantial first-pass metabolism occurs in the liver, where 21 

### biotransformation into urea and glutamine occur. I n normal circumstances, ammonia exists in 22 

### the blood as ammonium ion (NH 4 + 

### ). Ammonia or ammonium ion reaching the tissues is utilized 23 

### for glutamate production, which participates in tra nsamination and other reactions. The principal 24



### means of excretion of absorbed ammonia in mammals i s as urinary urea; minimal amounts are 25 

### excreted in the feces and in expired air. 26 

### As a liquid, ammonia is capable of burning the skin and causing permanent eye damage. 27 

### Case reports in humans following intentional or acc idental ingestion of household cleaning 28 

### products or ammonia smelling salts observed neurolo gical and gastrointestinal clinical symptoms 29 

### and burns along the digestive tract. As a gas, amm onia is capable of causing severe eye damage 30 

### and mild to severe pulmonary irritation and/or infl ammation (see human case studies and case 31 

### reports presented in Appendix C.2). Occupational e xposure studies and controlled human 32 

### exposure studies indicate respiratory symptoms (e.g ., coughing, wheezing, etc.), altered lung 33 

### function, and respiratory irritation following inha lation exposure to ammonia. Animal studies 34 

### also reported respiratory irritation and inflammati on following ammonia inhalation. 35 

### As discussed in Section 4.7, under EPA’s Guidelines for Carcinogen Risk Assessment 36 

### (U.S. EPA, 2005a), there is “inadequate information to assess the carcinogenic potential” of 37 

### ammonia. A series of studies provided limited evid ence that ammonia may act as a cancer 38

### 65 DRAFT - DO NOT CITE OR QUOTE

promoter, and positive responses in a few genotoxic ity studies have been reported, including one 1 

### study in fertilizer factory workers. 2

3



### 6.2. DOSE RESPONSE 4



### 6.2.1. Noncancer/Oral 5



### The available oral data for ammonia were considere d insufficient to support reference 6 

### value derivation. Therefore, an RfD for ammonia wa s not derived. 7

8



### 6.2.2. Noncancer/Inhalation 9 

### The RfC for ammonia of 0.3 mg/m 3 

### was derived using a duration-adjusted NOAEL of 10 

### 3.1 mg/m 3 

### as the POD based on a lack of respiratory symptoms and the absence of changes in 11 

### lung function at this concentration in occupational ly exposed workers at a soda ash plant 12 

### (Holness et al., 1989). To derive the RfC, the POD was divided by a total UF of 10 to account 13 

### for human interindividual variability. The default human variability factor was applied because 14 

### of the lack of quantitative information to assess t oxicokinetic or toxicodynamic differences in the 15 

### range of susceptibilities to ammonia in the human p opulation. 16 

### A confidence level of high, medium, or low is assig ned to the study used to derive the 17 

### RfC, the overall database, and the RfC itself, as d escribed in Section 4.3.9.2 of EPA’s Methods 18 

### for Derivation of Inhalation Reference Concentratio ns and Application of Inhalation Dosimetry 19 

### (U.S. EPA, 1994b). The overall confidence in the R fC for ammonia is medium. Confidence in 20 

### the principal study (Holness et al., 1989) is mediu m. The design, conduct, and reporting of this 21 

### occupational exposure study were adequate, but the study was limited by a small sample size and 22 

### by the fact that workplace ammonia concentrations t o which the study population was exposed 23



### were below those associated with ammonia-related ef fects (i.e., only a NOAEL was identified). 24 

### However, this study is supported in the context of the entire database, including the NOAEL and 25 

### LOAEL values identified in the Rahman et al. (2007) occupational exposure study, multiple 26 

### studies of acute ammonia exposure in volunteers, an d the available subchronic and chronic 27



### inhalation data from animals. Confidence in the da tabase is medium. The inhalation ammonia 28 

### database includes limited studies of reproductive t oxicity and no studies of developmental 29 

### toxicity; however, reproductive, developmental, and other systemic effects are not expected at 30



### the RfC because it is well documented that ammonia is endogenously produced in humans and 31 

### animals, because ammonia concentrations in blood ar e homeostatically regulated to remain at 32 

### low levels, and because ammonia concentrations in a ir at the POD are not expected to alter 33 

### homeostasis. Reflecting medium confidence in the p rincipal study and medium confidence in 34 

### the database, the overall confidence in the RfC is medium. 35

36

### 66 DRAFT - DO NOT CITE OR QUOTE

6.2.3. Cancer 1



### No cancer assessment was conducted for ammonia due to the lack of adequate 2 

### carcinogenicity data in humans or animals. 3

### 67 DRAFT - DO NOT CITE OR QUOTE 7. REFERENCES

Abramovicz, I. (1924) Ocular injury caused by liqui d ammonia. Br J Ophthalmol 9(5):241–242.

Ahboucha, S; Araqi, F; Layrargues, GP; et al. (2005 ) Differential effects of ammonia on the benzodiaze pine modulatory site on the GABA-A receptor complex of h uman brain. Neurochem Int 47(1–2):58–63.

AIChE (1999) Ammonia H3N. Physical and thermodynami c properties of pure chemicals. American Institute of Chemical Engineers, Design Institute for Physical P roperty Data. Philadelphia, PA: Taylor and Francis.

Albrecht, J. (1996) Astrocytes and ammonia neurotox icity. 2nd edition. New York, NY: CRC Press, pp. 1 37–153.

Albrecht, J; Norenberg, MD. (2006) Glutamine: a Tro jan horse in ammonia neurotoxicity. Hepatology 44( 4):788– 794.

Albrecht, J; Zielinska, M; Norenberg, MD. (2010) Gl utamine as a mediator of ammonia neurotoxicity: a c ritical appraisal. Biochem Pharmacol 80(9):1303–1308.

Ali, BA. (2001) Pulmonary function of workers expos ed to ammonia: a study in the eastern province of S audi Arabia. Int J Occup Environ Health 7(1):19–22.

Altmann, L; Berresheim, H; Kruell, H; et al. (2006) Odor and sensory irritation of ammonia measured in humans. Naunyn-Schmiedebergs Arch Pharmakol 372(Suppl. 1):9 9.

Amshel, CE; Fealk, MH; Phillips, BJ; et al. (2000) Anhydrous ammonia burns case report and review of t he literature. Burns 26(5):493–497.

Anderson, DP; Beard, CW; Hanson, RP. (1964) The adv erse effects of ammonia on chickens including resis tance to infection with Newcastle disease virus. Avian Dis 8:369–379.

Andersson, BO. (1991) Lithuanian ammonia accident, March 20th 1989. Institution of Chemical Engineers Symposium Series 124:15–17.

Appelman, LM; ten Berge, WF; Reuzel, PGJ. (1982) Ac ute inhalation toxicity of ammonia in rats with var iable exposure periods. Am Ind Hyg Assoc J 43(9):662–665

.

Arwood, R; Hammond, J; Gillon Ward, G. (1985) Ammon ia inhalation. J Trauma 25(5):444–447.

ATSDR (Agency for Toxic Substances and Disease Regi stry). (2004) Toxicological profile for ammonia (up date). Atlanta, GA. Available online at http://www.atsdr.c dc.gov/ (accessed August 20, 2009).

Auerbach, C; Robson, JM. (1947) XXXIII. Test of che mical substances for mutagenic action. Proc R Soc Edinb [Nat Environ] 62:284–291.

Bachmann, C. (2002) Mechanisms of hyperammonemia. Clin Chemistry Lab Med 40(7):653–662.

Bai, G. (2001) Ammonia induces the mitochondrial pe rmeability transition in primary cultures of rat as trocytes. J Neurosci Res 66(5):981–991.

Ballal, SG; Ali, BA; Albar, AA; et al. (1998) Bronc hial asthma in two chemical fertilizer producing fa ctories in eastern Saudi Arabia. Int J Tuberc Lung Dis 2(4):3 30–335.

Barrow, CS; Steinhagen, WH. (1980) NH 3 concentrations in the expired air of the rat: impo rtance to inhalation toxicology. Toxicol Appl Pharmacol 53:116–121.

Basile, AS. (2002) Direct and indirect enhancement of GABAergic neurotransmission by ammonia: implicat ions for the pathogenesis of hyperammonemic syndromes. Neur ochem Int 41:115–122.

### 68 DRAFT - DO NOT CITE OR QUOTE

Beare, JD; Wilson, RS; Marsh, RJ. (1988) Ammonia bu rns of the eye: old weapon in new hands. Br Med J 296:590.

Bell, AW; Kennaugh, JM; et al. (1989) Uptake of ami no acids and ammonia at midgestation by the fetal l amb. Q J Exp Physiol 74:635-643.

Bento, LM; Fagian, MM; Vercesi, AE; et al. (2007) E ffects of NH 4 Cl-induced systemic metabolic acidosis on kidney mitochondrial coupling and calcium transport in rats. Nephrol Dial Transplant 22(10):2817–2823

.

Bernstein, IL; Bernstein, DL. (1989) Reactive airwa ys disease syndrome (RADS) after exposure to toxic ammonia fumes. J Allergy Clin Immunol 83:173.

Betterton, EA. (1992) Henry's law constants of solu ble and moderately soluble organic gases: effects o n aqueous phase chemistry. Tucson, AZ: John Wiley & Sons, pp. 1–50.

Bishop, JM; Verlander, JW; Lee, HW; et al. (2010) R ole of the Rhesus glycoprotein, Rh B glycoprotein, in renal ammonia excretion. Am J Physiol Renal Physiol 299( 5):F1065–F1077.

Bloom, GR; Suhail, F; Hopkins-Price, P; et al. (200 8) Acute anhydrous ammonia injury from accidents du ring illicit methamphetamine production. Burns 34(5):713–718.

Bodega, G; Suarez, I; Paniagua, C; et al. (2007) Ef fect of ammonia, glutamine, and serum on calcineuri n, p38MAPK-diP, GADD153/CHOP10, and CNTF in primary ra t astrocyte cultures. Brain Res 1175:126–133.

Boshier, PR; Marczin, N; Hanna, GB. (2010) Repeatab ility of the measurement of exhaled volatile metabo lites using selected ion flow tube mass spectrometry. J Am Soc Mass Spectrom 21(6):1070–1074.

Boyano-Adanez, MC; Bodega, G; Barrios, V; et al. (1 996) Response of rat cerebral somatostatinergic sys tem to a high ammonia diet. Neurochem Res 29(5):469–476.

Boyd, EM; MacLachlan, ML; Perry, WF. (1944) Experim ental ammonia gas poisoning in rabbits and cats. J Ind Hyg Toxicol 26(1):29–34.

Brautbar, N; Wu, MP; Richter, ED. (2003) Chronic am monia inhalation and interstitial pulmonary fibrosi s: a case report and review of the literature. Arch Environ Health 58(9):592–596.

Broderson, JR; Lindsey, JR; Crawford, JE. (1976) Th e role of environmental ammonia in respiratory myco plasmosis of rats. Am J Pathol 85:115–130.

Brown, RH; Duda, GD; Korkes, S; et al. (1957) A col orimetric micromethod for determination of ammonia; the ammonia content of rat tissues and human plasma. A rch Biochem Biophys 66:301–309.

Buckley, LA; Jiang, XJ; James, RA; et al. (1984) Re spiratory tract lesions induced by sensory irritant s at the RD 50 concentration. Toxicol Appl Pharmacol 74:417–429.

Bugge, M; Bengtsson, F; Nobin, A. (1988) Serotonin metabolism in the rat brain following ammonia admin istration. In: Soeters, PB; Wilson, JHP; Meijer, AJ; et al., eds. Advances in ammonia metabolism and hepatic encephalopathy. Amsterdam: Excerpta Medica, pp. 454 –461.

Burns, TR; Greenberg, SD; Mace, ML; et al. (1985) U ltrastructure of acute ammonia toxicity in the huma n lung. Am J Forensic Med Pathol 6:204–210.

Caplin, M. (1941) Ammonia-gas poisoning. Forty-seve n cases in a London smelter. Lancet 2:95–96.

Castell, DO; Moore, EW. (1971) Ammonia absorption f rom the human colon. Gastroenterology 60:33–42.

Chastre, A; Jiang, W; Desjardins, P; et al. (2010) Ammonia and proinflammatory cytokines modify expres sion of genes coding for astrocytic proteins implicated in brain edema in acute liver failure. Metab Brain Di s 25(1):17–21.

### 69 DRAFT - DO NOT CITE OR QUOTE

Chaung, H; Hsia, L; Liu, S. (2008) The effects of v itamin A supplementation on the production of hyper sensitive inflammatory mediators of ammonia-induced airways o f pigs. Food Agric Immunol 19(4):283–291.

ChemIDplus. (2009) Ammonia. Bethesda, MD: U.S. Nat ional Library of Medicine. Available online at http://sis.nlm.nih.gov/chemical.html (accessed on A ugust 17, 2009).

Choudat, D; Goehen, M; Korobaeff, M; et al. (1994) Respiratory symptoms and bronchial reactivity among pig and dairy farmers. Scand J Work Environ Health 20(1):4 8–54.

Christesen, HB. (1995) Prediction of complications following caustic ingestion in adults. Clin Otolar yngol 20(3):272–278.

Clark, EC; Nath, KA; Hostetter, MK; et al. (1990) R ole of ammonia in tubulointerstital injury. Miner Electrolyte Metab 16(5):315–321.

Close, LG; Catlin, FI; Cohn, AM. (1980) Acute and c hronic effects of ammonia burns of the respiratory tract. Arch Otolaryngol 106:151–158.

Cole, TJ; Cotes, JE; Johnson, GR. (1977) Ventilatio n, cardiac frequency and pattern of breathing durin g exercise in men exposed to O-chlorobenzylidene malonitrile (CS) and ammonia gas in low concentrations. Q J Exp Ph ysiol 62:341–351.

Conn, HO. (1972) Studies of the source and signific ance of blood ammonia. IV. Early ammonia peaks afte r ingestion of ammonia salts. Yale J Biol Med 45:543 –549.

Coon, RA; Jones, RA; Jenkins, LJ; et al. (1970) Ani mal inhalation studies on ammonia, ethylene, glycol

, formaldehyde, dimethylamine and ethanol. Toxicol A ppl Pharmacol 16:646–655.

Córdoba, J. (1998) Chronic hyponatremia exacerbates ammonia-induced brain edema in rats after portacav al anastomosis. J Hepatol 29(4):589–594.

Cormier, Y; Israel-Assayag, E; Racine, G; et al. (2 000) Farming practices and the respiratory health r isks of swine confinement buildings. Eur Respir J 15(3):560–565.

Counturier, Y; Barbotin, M; Bobin, P; et al. (1971) Apropos of three cases of toxic lung caused by vap ors of ammonia and hydrogen sulfide. Bull Soc Med d'Afriq ue Noire de Langue Francaise 16(2):250–252.

Crook, B; Robertson, JF; Travers Glass, SA; et al. (1991) Airborne dust, ammonia, microorganisms, and antigens in pig confinement houses and the respiratory health o f exposed farm workers. Am Ind Hyg Assoc J 52(7):2 71–279.

Curtis, SE; Anderson, CR; Simon, J; et al. (1975) E ffects of aerial ammonia, hydrogen sulfide and swin e-house dust on rate of gain and respiratory-tract structure in swine. J Anim Sci 41(3):735–739.

da Fonseca-Wollheim, F. (1995) The influence of pH and various anions on the distribution of NH 4 + in human blood. Eur J Clin Chem Clin Biochem 33(5):289–294

.

Dalhamn, T. (1963) Effect of ammonia alone and comb ined with carbon particles on ciliary activity in t he rabbit trachea in vivo, with studies of the absorption cap acity of the nasal cavity. Int J Air Water Pollut 7:531–539.

Davies, BMA; Yudkin, J. (1952) Studies in biochemic al adaptation. The origin of urinary ammonia as ind icated by the effect of chronic acidosis and alkalosis on som e renal enzymes in the rat. Biochem J 52:407–412.

Davies, S; Špan ě l, P; Smith, D.l. (1997) Quantitative analysis of a mmonia on the breath of patients in end-stage renal failure. Kidney Int 52:223–228.

Dean, JA (1985) Lange's handbook of chemistry. New York, NY: McGraw-Hill Book Co., pp. 10-3, 10-23.

de la Hoz, RE; Schlueter, DP; Rom, WN. (1996) Chron ic lung disease secondary to ammonia inhalation inj ury: a report on three cases. Am J Ind Med 29(2):209–214.

### 70 DRAFT - DO NOT CITE OR QUOTE

Demerec, M; Bertau, G; Flint, J. (1951) A survey of chemicals for mutagenic action on E. coli. Am Nat 85:119– 136.

DeSanto, JT; Nagomi, W; Liechty, EA; Lemons, JA. (1 993). Blood ammonia concentration in cord blood dur ing pregnancy. Early Hum Dev 33(1):1-8.

Diack, C; Bois, FY. (2005) Pharmacokinetic-pharmaco dynamic models for categorical toxicity data. Regu l Toxicol Pharmacol 41(1):55–65.

Diekman, MA; Scheidt, AB; Sutton, AL; et al. (1993) Growth and reproductive performance, during exposu re to ammonia, of gilts afflicted with pneumonia and atro phic rhinitis. Am J Vet Res 54(12):2128–2131.

Dilli, D; Bostanci, I; Tiras, U; et al. (2005) A no n-accidental poisoning with ammonia in adolescence. Child Care Health Dev 31(6):737–739.

Diskin, A et al. (2003) Time variation of ammonia, acetone, isoprene and ethanol in breath: a quantita tive SIFT-MS study over 30 days. Physiol Meas 24: 107-119.

Dodd, KT; Gross, DR. (1980) Ammonia inhalation toxi city in cats: a study of acute and chronic respirat ory dysfunction. Arch Environ Health 35:6–14.

Doig, PA; Willoughby, RA. (1971) Response of swine to atmospheric ammonia and organic dust. J Am Vet Med Assoc 159:1353–1361.

Dolinska, M; Hilgier, W; Albrecht, J. (1996) Ammoni a stimulates glutamine uptake to the cerebral non-s ynaptic mitochondria of the rat. Neurosci Lett 213(1):45–4 8.

Done, SH; Chennells, DJ; Gresham, AC; et al. (2005) Clinical and pathological responses of weaned pigs to atmospheric ammonia and dust. Vet Rec 157(3):71–80

.

Donham, KJ; Reynolds, SJ; Whitten, P; et al. (1995) Respiratory dysfunction in swine production facili ty workers: dose-response relationships of environmental exposu res and pulmonary functions. Am J Ind Med 27(3):40 5–418.

Donham, KJ; Cumro, D; Reynolds, SJ; et al. (2000) D ose-response relationships between occupational aer osol exposures and cross-shift declines of lung function in poultry workers: recommendations for exposure l imits. J Occup Environ Med 42(3):260–269.

Douglas, RB; Coe, JE. (1987) The relative sensitivi ty of the human eye and lung to irritant gases. An nal Occup Hyg 31(2):265–267.

Drummond, JG; Curtis, SE; Simon, J; et al. (1980) E ffects of aerial ammonia on growth and health of yo ung pigs. J Anim Sci 50(6):1085–1091.

Dworkin, MS; Patel, A; Fennell, M; et al. (2004) An outbreak of ammonia poisoning from chicken tenders served in a school lunch. J Food Prot 67(6):1299–1302.

Eggeman, T (2001) Ammonia. In: Kirk-Othmer encyclo pedia of chemical technology. New York, NY: John Wi ley & Sons, Inc, pp. 678–710.

Egle, JL. (1973) Retention of inhaled acetone and a mmonia in the dog. Am Ind Hyg Assoc J 34:533–539.

Elfman, L; Riihimaki, M; Pringle, J; et al. (2009) Influence of horse stable environment on human airw ays. J Occup Med Toxicol 4:10.

Fazekas, IG. (1939) Experimental suprarenal hypertr ophy induced by ammonia. Endokrinologie 21:315–337

.

Felipo, V; Butterworth, RF. (2002) Neurobiology of ammonia. Prog Neurobiol 67:259–279.

### 71 DRAFT - DO NOT CITE OR QUOTE

Felipo, V; Hermenegildo, C; Montoliu, C; et al. (19 98) Neurotoxicity of ammonia and glutamate: molecul ar mechanisms and prevention. Neurotoxicology 19(4–5) :675–681.

Felipo, V; Monfort, P; Fernandez-Marticorena, I; et al. (2000) Activation of NMDA receptors in rat bra in in vivo following acute ammonia intoxication. Characterizat ion by in vivo brain microdialysis. Eur J Neurosci 12(Suppl 11):40.

Ferguson, WS; Koch, WC; Webster, LB; et al. (1977) Human physiological response and adaption to ammoni a. J Occup Med 19:319–326.

Ferris, BG. (1978) Epidemiology Standardization Pro ject. Am Rev Respir Dis 118:11–31.

Fischer, ML; Littlejohn, D; Lunden, MM; et al. (200 3) Automated measurements of ammonia and nitric aci d in indoor and outdoor air. Environ Sci Technol 37(10) :2114-2119.

Flessner, MF; Wall, SM; Knepper, MA. (1992) Ammoniu m and bicarbonate transport in rat outer medullary collecting ducts. Am J Phyiol 262(1 Pt 2):F1–F7.

Flury, KE; Dines, DE; Rodarte, JR; et al. (1983) Ai rway obstruction due to inhalation of ammonia. May o Clin Proc 58:389–393.

Gaafar, H; Girgis, R; Hussein, M; et al. (1992) The effect of ammonia on the respiratory nasal mucosa of mice. A histological and histochemical study. Acta Oto Lar yngologica 112(2):339–342.

Gamble, MR; Clough, G. (1976) Ammonia build-up in a nimal boxes and its effect on rat tracheal epitheli um. Lab Anim. 10:93–104.

Garcia, MV; Lopez-Mediavilla, C; Juanes de la Pena, MC; et al. (2003) Tolerance of neonatal rat brain to acute hyperammonemia. Brain Res 973(1):31–38.

Gay, WMB; Crane, CW; Stone, WD. (1969) The metaboli sm of ammonia in liver disease: a comparison of uri nary data following oral and intravenous loading of [ 15 N] ammonium lactate. Clin Sci 37:815–823.

George, A; Bang, RL; Lari, AR; et al. (2000) Liquid ammonia injury. Burns 26(4):409–413.

Gilbert, GJ. (1988) Acute ammonia intoxication 37 y ears after ureterosigmoidostomy. South Med J 81(11 ):1443– 1445.

Giordano, G; Sanchez-Perez, AM; Burgal, M; et al. ( 2005) Chronic exposure to ammonia induces isoform-s elective alterations in the intracellular distribution and N MDA receptor-mediated translocation of protein kina se C in cerebellar neurons in culture. J Neurochem 92(1):1 43–157.

Giroux, M; Bremont, F; Salles, JP; et al. (2002) Ex haled NH 3 and excreted NH 4 + in children in unpolluted or urban environments. Environ Int 28(3):197–202.

Gomzi, M; Šari ć , M. (1997) Respiratory impairment among children l iving in the vicinity of a fertilizer plant. Int Arch Occup Environ Health 70(5):314–320.

Gorg, B; Qvartskhava, N; Keitel, V; et al. (2008) A mmonia induces RNA oxidation in cultured astrocytes and brain in vivo. Hepatology 48(2):567–579.

Gottschalk, S; Zwingmann, C. (2009) Altered fatty a cid metabolism and composition in cultured astrocyt es under hyperammonemic conditions. J Neurochem 109 (Suppl 1):258–264.

Green, AR; Wathes, CM; Demmers, TG; et al. (2008) D evelopment and application of a novel environmental preference chamber for assessing responses of labor atory mice to atmospheric ammonia. J Am Assoc Lab Anim Sci 47(2):49–56.

### 72 DRAFT - DO NOT CITE OR QUOTE

Gustin, P; Urbain, B; Prouvost, JF; et al. (1994) E ffects of atmospheric ammonia on pulmonary hemodyna mics and vascular permeability in pigs: interaction with end otoxins. Toxicol Appl Pharmacol 125(1):17–26.

Guyton, AC (1981) The body fluids and kidneys. In: Textbook of medical physiology. 6th edition. Phil adelphia, PA: WB Saunders Company, pp. 456–458, 889.

Hamid, HA; El-Gazzar, RM. (1996) Effect of occupati onal exposure to ammonia on enzymatic activities of catalase and mono amine oxidase. J Egypt Public Health Asso c 71(5-6):465–475.

Han, KH; Croker, BP; Clapp, WL; et al. (2006) Expre ssion of the ammonia transporter, Rh C glycoprotein , in normal and neoplastic human kidney. J Am Soc Nephr ol 17(10):2670–2679.

Handlogten, ME; Hong, SP; Westhoff, CM; et al. (200 5) Apical ammonia transport by the mouse inner medu llary collecting duct cell (mIMCD-3). Am J Physiol Renal Physiol 289(2):F347–358.

Hatton, DV; Leach, CS; Beaudet, AL; et al. (1979) C ollagen breakdown and ammonia inhalation. Arch Env iron Health 34:83–86.

Hauguel, S; Challier, JC; et al. (1983) Metabolism of the human placenta perfused in vitro: glucose tr ansfer and utilization, O2 consumption, lactate and ammonia pr oduction. Pediatr Res 17:729-732.

Hawkins, RA; Miller, AL; Nielsen, RC; et al. (1973) The acute action of ammonia on rat brain metabolis m in vivo. Biochem J 134:1001–1008.

Heederik, D; van Zwieten, R; Brouwer, R. (1990) Acr oss-shift lung function changes among pig farmers. Am J Ind Med 17(1):57–58.

Heifer, U. (1971) Casuistic contribution to acute l ethal inhalation poisoning by ammonia. Lebensversi cher Med 22:60–62. (German)

Helmers, S; Top, FH; Knapp, LW. (1971) Ammonia inju ries in agriculture. J Iowa Med Soc 61(5):271–280.

Hermenegildo, C; Marcaida, G; Montoliu, C; et al. ( 1996) NMDA receptor antagonists prevent acute ammon ia toxicity in mice. Neurochem Res 21(10):1237–1244.

Hertz, L; Kala, G. (2007) Energy metabolism in brai n cells: effects of elevated ammonia concentrations . Metab Brain Dis 22(3-4):199–218.

Highman, VN. (1969) Early rise in intraocular press ure after ammonia burns. Br Med J. 1(5640):359–360

.

Hilado, CJ; Casey, CJ; Furst, A. (1977) Effect of a mmonia on Swiss albino mice. J Combust Toxicol 4:3 85–388.

Hoeffler, HB; Schweppe, HI; Greenberg, SD. (1982) B ronchiectasis following pulmonary ammonia burn. Ar ch Pathol Lab Med 106:686–687.

Holness, DL; Purdham, JT; Nethercott, JR. (1989) Ac ute and chronic respiratory effects of occupational exposure to ammonia. Am Ind Hyg Assoc J 50(12):646–650.

Holzman, IR; Phillips, AF; Battaglia, FC. (1979) Gl ucose metabolism, lactate, ammonia production by th e human placenta in vitro. Pediatr Res 13:117-120.

HSDB (Hazardous Substances Data Bank). (2009) Ammon ia. National Library of Medicine. Available online at http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB (accessed August 17, 2009).

Huizenga, JR; Tangerman, A; Gips, CH. (1994) Determ ination of ammonia in biological fluids. Ann Clin Biochem 31(6):529–543.

Igarashi, M; Kitada, Y; Yoshiyama, H; et al. (2001) Ammonia as an accelerator of tumor necrosis factor alphainduced apoptosis of gastric epithelial cells in Helicobacter pylori infection. Infect Immun 69(2):816–821.

### 73 DRAFT - DO NOT CITE OR QUOTE

Ihrig, A; Hoffman, J; Triebig, G. (2006) Examinatio n of the influence of personal traits and habituati on on the reporting of complaints at experimental exposure to ammonia. Int Arch Occup Environ Health 79(4):332– 338.

Izumi, Y; Izumi, M; Matsukawa, M; et al. (2005) Amm onia-mediated LTP inhibition: effects of NMDA recep tor antagonists and L-carnitine. Neurobiol Dis 20(2):6 15–624.

Jalan, R; Wright, G; Davies, NA; et al. (2007) L-Or nithine phenylacetate (OP): a novel treatment for hyperammonemia and hepatic encephalopathy. Med Hyp otheses 69(5):1064–1069.

Jarudi, MD; Golden, MD. (1973) Ammonia eye injuries . J Iowa Med Soc 63:260–263.

Jayakumar, AR; Panickar, KS; Murthy Ch, R; et al. ( 2006a) Oxidative stress and mitogen-activated prote in kinase phosphorylation mediate ammonia-induced cell swelli ng and glutamate uptake inhibition in cultured astr ocytes. J Neurosci 26(18):4774–4784.

Jayakumar, AR; Rao, KV; Murthy Ch, R; et al. (2006b ) Glutamine in the mechanism of ammonia-induced ast rocyte swelling. Neurochem Int 48(6–7):623–628.

Jayakumar, AR; Liu, M; Moriyama, M; et al. (2008) N a-K-Cl cotransporter-1 in the mechanism of ammoniainduced astrocyte swelling. J Biol Chem 283(49):33 874–33882.

Jayakumar, AR; Rama Rao, KV; Tong, XY; et al. (2009 ) Calcium in the mechanism of ammonia-induced astro cyte swelling. J Neurochem 109(Suppl 1):252–257.

Jeffers, LJ. (1988) Hepatic encephalopathy and orot ic aciduria associated with hepatocellular carcinom a in a noncirrhotic liver. Hepatology 8(1):78–81.

Johnson, RL; Gilbert, M; et al. (1986) Uterine meta bolism of the pregnant rabbit under chronic steady- state conditions. Am J Obstet Gynecol 154:1146-51.

Johnson, DR Bedick, CR, Clark NN, McKain DL Jr. (20 09) Design and testing of an independently controll ed urea SCR retrofit system for the reduction of NOx emissi ons from marine diesels. Environ Sci Technol 43(10) : 3959- 3963.

Jones, EA. (2002) Ammonia, the GABA neurotransmitte r system, and hepatic encephalopathy. Metab Brain Dis 17(4):275–281.

J όź wik, M; Teng, C; Meschia, G; et al. (1999) Contribu tion of branched-chain amino acids to uteroplacenta l ammonia production in sheep. Biol Repro 61:792-796.

J όź wik, M; Pietrzycki, B; Chojnowski, M; et al. (2005) Maternal and fetal blood ammonia concentrations in normal term human pregnancies. Biol Neonate 87(1):38–43.

Kala, G; Hertz, L. (2005) Ammonia effects on pyruva te/lactate production in astrocytes--interaction wi th glutamate. Neurochem Int 47(1–2):4–12.

Kalandarov, S; Bychkov, VP; Frenkel, ID; et al. (19 84) Effect of an increased ammonia content in an en closed atmosphere on the adrenocortical system in man. Ko sm Bioloi Aviak Med 18:75–77.

Kanamori, K; Ross, BD; Chung, JC; et al. (1996) Sev erity of hyperammonemic encephalopathy correlates w ith brain ammonia level and saturation of glutamine syn thetase in vivo. J Neurochem 67(4):1584–1594.

Kane, LE; Barrow, CS; Alarie, Y. (1979) A short-ter m test to predict acceptable levels of exposure to airborne sensory irritants. Am Ind Hyg Assoc J 40:207–229.

Kapeghian, JC; Jones, AB; Waters, IW. (1985) Effect s of ammonia on selected hepatic microsomal enzyme activity in mice. Bull Environ Contam Toxicol 35:15–22.

### 74 DRAFT - DO NOT CITE OR QUOTE

Kapeghian, JC; Mincer, HH; Jones, AL; et al. (1982) Acute inhalation toxicity of ammonia in mice. Bul l Environ Contam Toxicol 29:371–378.

Kass, I; Zamel, N; Dobry, CA; et al. (1972) Bronchi ectasis following ammonia burns of the respiratory tract. Chest 62(3):282–285.

Katayama, K. (2004) Ammonia metabolism and hepatic encephalopathy. Hepatol Res 30S:73–80.

Kawano, S; et al. (1991) Chronic effect of intragas tric ammonia on gastric mucosal structures in rats. Digestive Disorders and Sciences 36(1):33-38.

Kearney, DJ; Hubbard, T; Putnam, D. (2002) Breath a mmonia measurement in Helicobacter pylori infection. Dig Dis Sci 47(11):2523–2530.

Keiding, S; Sorensen, M; Bender, D; et al. (2006) B rain metabolism of 13 N-ammonia during acute hepatic encephalopathy in cirrhosis measured by positron em ission tomography. Hepatology 43(1):42–50.

Keiding, S; Sorensen, M; Munk, OL; et al. (2010) Hu man 13 N-ammonia PET studies: the importance of measuring

13 N-ammonia metabolites in blood. Metab Brain Dis 25 (1):49–56.

Kerstein, MD; Schaffzin, DM; Hughes, WB; et al. (20 01) Acute management of exposure to liquid ammonia. Mil Med 166(10):913–914.

Kitano, T; Matsumura, S; Seki, T; et al. (2004) Cha racterization of N-methyl-D-aspartate receptor subu nits involved in acute ammonia toxicity. Neurochem Int 44(2):83– 90.

Klein, J; Olson, KR; McKinney, HE. (1985) Caustic i njury from household ammonia. Am J Emerg Med 3:320

.

Klejman, A; Wegrzynowicz, M; Szatmari, EM; et al. ( 2005) Mechanisms of ammonia-induced cell death in r at cortical neurons: roles of NMDA receptors and gluta thione. Neurochem Int 47(1–2):51–57.

Klendshoj, NC; Rejent, TA. (1966) Tissue levels of some poisoning agents less frequently encountered. J Forensic Sci 11(1):75–80.

Konopacka, A; Fresko, I; Piaskowski, S; et al. (200 6) Ammonia affects the activity and expression of s oluble and particulate GC in cultured rat astrocytes. Neuroch em Int 48(6–7):553–558.

Konopacka, A; Zielinska, M; Albrecht, J. (2008) Amm onia inhibits the C-type natriuretic peptide-depend ent cyclic GMP synthesis and calcium accumulation in a rat bra in endothelial cell line. Neurochem Int 52(6):1160 –1166.

Kosenko, E; Kaminsky, YG; Felipo, V; et al. (1993) Chronic hyperammonemia prevents changes in brain en ergy and ammonia metabolites induced by acute ammonium i ntoxication. Biochim Biophysica Acta 1180(3):321–3 26.

Kosenko, E; Kaminsky, Y; Grau, E; et al. (1994) Bra in ATP depletion induced by acute ammonia intoxicat ion in rats is mediated by activation of the NMDA receptor and Na+, K(+)-ATPase. J Neurochem 63(6):2172–2178

.

Kosenko, E; Kaminiski, Y; Lopata, O; et al. (1999) Blocking NMDA receptors prevents the oxidative stre ss induced by acute ammonia intoxication. Free Radic Biol Med 22(11/12):1369–1374.

Kosenko, E; Venediktova, N; Kaminsky, Y; et al. (20 03) Sources of oxygen radicals in brain in acute am monia intoxication in vivo. Brain Res 981(1–2):193–200.

Kosenko, E; Montoliu, C; Giordano, G; et al. (2004) Acute ammonia intoxication induces an NMDA recepto rmediated increase in poly(ADP-ribose) polymerase le vel and NAD metabolism in nuclei of rat brain cells . J Neurochem 89(5):1101–1110.

Kosenko, E; Kaminsky, Y; Solomadin, I; et al. (2007 ) Acute ammonia neurotoxicity in vivo involves incr ease in cytoplasmic protein P53 without alterations in othe r markers of apoptosis. J Neurosci Res 85(11):2491 –2499.

### 75 DRAFT - DO NOT CITE OR QUOTE

Kurtz, I. (1991) Role of ammonia in the induction o f renal hypertrophy. Am J Kidney Dis 17(6):650–653

.

Lalic, H; Djindjic-Pavicic, M; Kukuljan, M. (2009) Ammonia intoxication on workplace-case report and a review of literature. Coll Antropol 33(3):945–949.

Landahl, HD; Hermann, RG. (1950) Retention of vapor s and gases in the human nose and lung. Arch Ind H yg Occup Med 1:36–45.

Larson, T. (1980) The chemical neutralization of in haled sulfuric acid aerosol. Am J Ind Med 1:449–45 2.

Larson, TV; Covert, DS; Frank, R; et al. (1977) Amm onia in the human airways: neutralization of inspir ed acid sulfate aerosols. Science 197:161–163.

Latenser, BA; Lucktong, TA. (2000) Anhydrous ammoni a burns: case presentation and literature review. J Burn Care Rehabil 21(1 PT 1):40–42.

Leduc, D; Gris, P; Lheureux, P; et al. (1992) Acute and long term respiratory damage following inhalat ion of ammonia. Thorax 47(9):755–757.

Lee, HS; Chan, CC; Tan, KT; et al. (1993) Burnisher 's asthma: a case due to ammonia from silverware po lishing. Singapore Med J 34(6):565–566.

Lee, HW; Verlander, JW; Bishop, JM; et al. (2009) C ollecting duct-specific Rh C glycoprotein deletion alters basal and acidosis-stimulated renal ammonia excretion. A m J Physiol Renal Physiol 296(6):F1364–F1375.

Lee, HW; Verlander, JW; Bishop, JM; et al. (2010) E ffect of intercalated cell-specific Rh C glycoprote in deletion on basal and metabolic acidosis-stimulated renal ammon ia excretion. Am J Physiol Renal Physiol 299(2):F3 69–F379.

Levy, DM; Divertie, MB; Litzow, TJ; et al. (1964) A mmonia burns of the face and respiratory tract. J Am Med Assoc 190:873–876.

Li, WL; Pauluhn, J. (2010) Comparative assessment o f the sensory irritation potency in mice and rats n ose-only exposed to ammonia in dry and humidified atmosphere s. Toxicology 276(2):135–142.

Lide, DR (2008) CRC handbook of chemistry and physi cs. 88 th edition. Boca Raton, FL: Taylor & Francis, pp. 4-4 6 to 4-48, 8-40.

Llansola, M; Erceg, S; Hernandez-Viadel, M; et al. (2002) Prevention of ammonia and glutamate neurotox icity by carnitine: molecular mechanisms. Metab Brain Dis 1 7(4):389–397.

Llansola, M; Rodrigo, R; Monfort, P; et al. (2007) NMDA receptors in hyperammonemia and hepatic encephalopathy. Metab Brain Dis 22(3-4):321–335.

Lobasov, M; Smirnov, F. (1934) On the nature of the action of chemical agents on mutational process in Drosophila melanogaster . II. The effect of ammonia on the occurrence of le thal transgressions. C R Seances Acad Sci Roum 3:177–178.

López-Aparicio, S; Smolík, J; Mašková, L; et al. (2 011) Relationship of indoor and outdoor air polluta nts in a naturally ventilated historical building envelope. Build Environ 46(7):1460-1468.

Lopez, G; Dean, BS; Krenzelok, EP. (1988) Oral expo sure to ammonia inhalants: a report of 8 cases. Ve t Hum Toxicol 30:350.

MacEwen, JD; Theodore, J; Vernot, EH (1970) Human e xposure to eel concentrations of monomethylhydrazin e. In: Wright-Patterson Air Force Base, OH: Aerospace Medi cal Research Laboratory, pp. 355–363.

Manninen, ATA; Savolainen, H. (1989) Effect of shor t-term ammonia inhalation on selected amino acids i n rat brain. Pharmacol Toxicol 64(3):244–246.

### 76 DRAFT - DO NOT CITE OR QUOTE

Manninen, A; Anttila, S; Savolainen, H. (1988) Rat metabolic adaptation to ammonia inhalation. Proc S oc Exp Biol Med 187(3):278–281.

Manolis, A. (1983) The diagnosis potential of breat h analysis. Clin Chem 29: 5–15.

Marcaida, G; Felipo, V; Hermenegildo, C; et al. (19 92) Acute ammonia toxicity is mediated by the NMDA type of glutamate receptors. FEBS Lett 296(1):67–68.

Mayan, MH; Merilan, CP. (1972) Effects of ammonia i nhalation on respiration rate of rabbits. J Anim S ci 34:448– 452.

McGuinness, R. (1969) Ammonia in the eye. Br Med J :575.

Mégraud, F; Neman-Simha, V; Brugmann, D. (1992) Fur ther evidence of the toxic effect of ammonia produc ed by Helicobacter pylori urease on human epithelial cells. Infect Immun 60 (5):1858–1863.

Melbostad, E; Eduard, W. (2001) Organic dust-relate d respiratory and eye irritation in Norwegian farme rs. Am J Ind Med 39(2):209–217.

Meschia, G; Battaglia, FC; et al. (1980) Utilizatio n of substrates by the ovine placenta in vivo. Fed Proc 39:245-249.

Millea, TP; Kucan, JO; Smoot, EC. (1989) Anhydrous ammonia injuries. J Burn Care Rehabil 10(5):448–45 3.

Miñana, MD; Felipo, V; Grisolia, S. (1989) Assembly and disassembly of brain tubulin is affected by hi gh ammonia levels. Neurochem Res 14(3):235–238.

Miñana, MD; Marcaida, G; Grisolia, S; et al. (1995) Prenatal exposure of rats to ammonia impairs NMDA receptor function and affords delayed protection against amm onia toxicity and glutamate neurotoxicity. J Neuro pathol Exp Neurol 54(5):644–650.

Monfort, P; Kosenko, E; Erceg, S; et al. (2002) Mol ecular mechanism of acute ammonia toxicity: role of NMDA receptors. Neurochem Int 41:95–102.

Monsen, ER. (1987) The Journal adopts SI units for clinical laboratory values. J Am Diet Assoc 87(3):3 56-378.

Montague, TJ; Macneil, AR. (1980) Mass ammonia inha lation. Chest 77:496–498.

Morgan, SE. (1997) Ammonia pipeline rupture: risk a ssessment to cattle. Vet Hum Toxicol 39(3):159161.

Mori, S; Kaneko, H; Mitsuma, T; et al. (1998) Impli cations of gastric topical bioactive peptides in am monia-induced acute gastric mucosal lesions in rats. Scand J Gas troenterol 33(4):386–393.

Morton, K. (2005) Fatal exposure to anhydrous ammon ia in a food manufacturing plant. J Occup Environ Hyg 2(6):D4–D43.

Mossberg, SM; Ross, G. (1967) Ammonia, movement in the small intestine: preferential transport by the ileum. J Clin Invest 46(4):490–498.

Mulder, JS; Van der Zalm, HO. (1967) Fatal case of ammonia poisoning. Tijdschrift voor Sociale Genees kunde 45:458–460.

Muntwyler, E; Iacobellis, M; Griffin, GE. (1956) Ki dney glutaminase and carbonic anhydrase activities and renal electrolyte excretion in rats. Am J Physiol 184:83 –90.

Murakami, M. (1995) Products of neutrophil metaboli sm increase ammonia-induced gastric mucosal damage. Dig Dis Sci 40(2):268–273.

Nagy, L; Kusstatscher, S; Hauschka, PV; et al. (199 6) Role of cysteine proteases and protease inhibito rs in gastric mucosal damage induced by ethanol or ammonia in the rat. J Clin Invest 98(4):1047–1054.

### 77 DRAFT - DO NOT CITE OR QUOTE

Nelson, DL; Cox, MM. (2008) Amino acid oxidation an d the production of urea. In: Lehninger Principles of Biochemistry. 5th edition. WH Freeman & Co., pp. 68 0, 683.

Neumann, R; Mehlhorn, G; Buchholz, I; et al. (1987) Experimental studies of the effect of chronic expo sure of suckling pigs to aerogenous toxic gas with ammonia of varying concentrations. Zentralbl Veterinaermed Reihe B 34:241–253.

NIOSH (National Institute for Occupational Safety a nd Health) (1979), National Institute for Occupatio nal Safety and Health: NIOSH Manual of Analytical Methods. Vol 5, 2ded. (DHEW/NISOSH Pub. No. 79-141). Washington

, D.C.: Government Printing Office,. p. S347.

Norenberg, MD; Rao, KVR; Jayakumar, AR. (2003) The mitochondrial permeability transition in ammonia neurotoxicity. Encephalopathy and nitrogen metabol ism in liver failure. International Symposium on H epatic Encephalopathy and Nitrogen Metabolism. Amsterdam, Netherlands, May 30-June 1, 2002, pp 267–285.

Norenberg, MD; Rama Rao, KV; Jayakumar, AR. (2004) Ammonia neurotoxicity and the mitochondrial permeability transition. J Bioenerg Biomembr 36(4) :303–307.

Norenberg, MD; Rao, KV; Jayakumar, AR. (2005) Mecha nisms of ammonia-induced astrocyte swelling. Metab Brain Dis 20(4):303–318.

Norenberg, MD; Jayakumar, AR; Rama Rao, KV; et al. (2007) New concepts in the mechanism of ammoniainduced astrocyte swelling. Metab Brain Dis 22(3-4 ):219–234.

Norenberg, MD; Rama Rao, KV; Jayakumar, AR. (2009a) Signaling factors in the mechanism of ammonia neurotoxicity. Metab Brain Dis 24(1):103–117.

Norenberg, MD; Rama Rao, KV; Jayakumar, AR; et al. (2009b) Synergism between ammonia and cytokines in the mechanism of astrocyte swelling. J Neurochem 108(S uppl. 1):117–118.

Norwood, DM; Wainman, T; Lioy, PJ; et al. (1992) Br eath ammonia depletion and its relevance to acidic aerosol exposure studies. Arch Environ Health 47(4):309–31 3.

NRC (National Research Council). (1983) Risk assess ment in the federal government: managing the proces s. Washington, DC: National Academy Press.

NSF (National Science Foundation). (1999) Environme ntal science and engineering for the 21st century. Interim report. National Science Board Task Force on the E nvironment.

O'Connor, EA; Parker, MO; McLeman, MA; et al. (2010 ) The impact of chronic environmental stressors on growing pigs, Sus scrofa (Part 1): stress physiology, production and play b ehaviour. Animal 4(11):1899–1909.

Ohara, K; Aoyama, M; Fujita, M; et al. (2009) Prolo nged exposure to ammonia increases extracellular gl utamate in cultured rat astrocytes. Neurosci Lett 462(2):109– 112.

Okada, M; Nakao, R; Hosoi, R; et al. (2009) In vivo monitoring of extracellular 13 N-glutamine derived from bloodborne 13 N-ammonia in rat striatum using microdialysis with radio-LC method. J Neurosci Methods 184(1):37–41.

O'Kane, GJ. (1983) Inhalation of ammonia vapour. A naesthesia 38:1208–1213.

O'Neil, MJ; Heckelman, PE; Koch, CB; et al. (2006) The Merck index. 14 th edition. Whitehouse Station, NJ: Merck & Co., Inc., pp. 83–89.

Osmond, AH; Tallents, CJ. (1968) Ammonia attacks. Br Med J 3:740.

Petrova, M; Diamond, J; Schuster, B; et al. (2008) Evaluation of trigeminal sensitivity to ammonia in asthmatics and healthy human volunteers. Inhal Toxicol 20(12):108 5–1092.

### 78 DRAFT - DO NOT CITE OR QUOTE

Phillips, CJ; Pines, MK; Latter, M; et al. (2010) T he physiological and behavioral responses of steers to gaseous ammonia in simulated long-distance transport by shi p. J Anim Sci 88(11):3579–3589.

Pinson, DM; Schoeb, TR; Lindsey, JR; et al. (1986) Evaluation by scoring and computerized morphometry of lesions of early mycoplasma pulmonis infection of a mmonia exposure in F344/N rats (erratum in Vet Path ol 23(6):785–786). Vet Pathol 23(5):550–555.

Pirjavec, A; Kovic, I; Lulic, I; et al. (2009) Mass ive anhydrous ammonia injury leading to lung transp lantation. J Trauma 67(4):E93–E97.

Pitts, RF. (1971) The role of ammonia production an d excretion in regulation of acid-base balance. N Engl J Med 284:32–38.

Preller, L; Heederik, D; Boleij, JSM; et al. (1995) Lung function and chronic respiratory symptoms of pig farmers: focus on exposure to endotoxins and ammonia and use of disinfectants. Occup Environ Med 52:654–660.

Price, S; Watts, JC. (2008) Ammonia gas incident. Anaesthesia 63(8):894–895.

Price, SK; Hughes, JE; Morrison, SC; et al. (1983) Fatal ammonia inhalation: a case report with autops y findings. S Afr Med J 64:952–955.

Prudhomme, JC; Shusterman, DJ; Blanc, PD. (1998) Ac ute-onset persistent olfactory deficit resulting fr om multiple overexposures to ammonia vapor at work. J Am Board Fam Pract 11(1):66–69.

Qin, C; Foreman, RD; Farber, JP. (2007a) Afferent p athway and neuromodulation of superficial and deepe r thoracic spinal neurons receiving noxious pulmonary inputs i n rats. Auton Neurosci 131(1–2):77–86.

Qin, C; Foreman, RD; Farber, JP. (2007b) Inhalation of a pulmonary irritant modulates activity of lumb osacral spinal neurons receiving colonic input in rats. Am J Physiol Regul Integr Comp Physiol 293(5):R2052–2 058.

Raabe, WA. (1989) Neurophysiology of ammonia intoxi cation. In: Butterworth, RF Pomier Layraegues, G; eds. Hepatic encephalopathy: pathophysiology and treatme nt. Clifton, NJ: Human Press, pp. 49–77.

Rabkin, R; Palathumpat, M; Tsao, T. (1993) Ammonium chloride alters renal tubular cell growth and prot ein turnover. Lab Invest 68(4):427–438.

Rahman, MH; Bratveit, M; Moen, BE. (2007) Exposure to ammonia and acute respiratory effects in a urea fertilizer factory. Int J Occup Environ Health 13(2):153–159.

Rama Rao, KV; Jayakumar, AR; Norenberg, DM. (2003) Ammonia neurotoxicity: role of the mitochondrial permeability transition. Metab Brain Dis 18(2):113 –127.

Rama Rao, KV; Jayakumar, AR; Norenberg, MD. (2005a) Role of oxidative stress in the ammonia-induced mitochondrial permeability transition in cultured a strocytes. Neurochem Int 47(1–2):31–38.

Rama Rao, KV; Panickar, KS; Jayakumar, AR; et al. ( 2005b) Astrocytes protect neurons from ammonia toxi city. Neurochem Res 30(10):1311–1318.

Rama Rao, KV; Jayakumar, AR; Tong, X; et al. (2010) Marked potentiation of cell swelling by cytokines in ammonia-sensitized cultured astrocytes. J Neuroinf lammation 7:66.

Read, AJ. (1982) Ionization constants of aqueous a mmonia from 25 to 250°C and to 2000 bar. J Solution Chem 11(9):649-664.

Reddy, PV; Rama Rao, KV; Norenberg, MD. (2009) Inhi bitors of the mitochondrial permeability transition reduce ammonia-induced cell swelling in cultured astrocyte s. J Neurosci Res 87(12):2677–2685.

Rejniuk, VL; Schafer, TV; Ovsep'yan, RV; et al. (20 07) Effect of atmospheric ammonia on mortality rate of rats with barbiturate intoxication. Bull Exp Biol Med 1 43(6):692–694.

### 79 DRAFT - DO NOT CITE OR QUOTE

Rejniuk, VL; Schafer, TV; Ivnitsky, JJ. (2008) Ammo nia potentiates the lethal effect of ethanol on rat s. Bull Exp Biol Med 145(6):741–743.

Remesar, K; Arola, L; Palou, A; et al. (1980) Activ ities of enzymes involved in amino acid metabolism in developing rat placenta. Eur J Biochem 110:289-293.

Renard, JJ; Calidonna, SE; Henley, MV. (2004) Fate of ammonia in the atmosphere- a review for applicab ility to hazardous releases. J Hazard Mater B108:29–60.

Reynolds, SJ; Donham, KJ; Whitten, P; et al. (1996) Longitudinal evaluation of dose-response relations hips for environmental exposures and pulmonary function in s wine production workers. Am J Ind Med 29(1):33–40

.

Richard, D; Bouley, G; Boudene, C. (1978a) Effects of continuous inhalation of ammonia in the rat and mouse. Bull Eur Physiopathol Respir 14:573–582. (French)

Richard, D; Jouany, JM; Boudene, C. (1978b) Acute t oxicity of ammonia gas in the rabbit by inhalation. C R Hebd Seances Acad Sci 287:375–378. (French)

Rose, C. (2006) Effect of ammonia on astrocytic glu tamate uptake/release mechanisms. J Neurochem 97(S uppl 1):11–15.

Rosenbaum, AM; Walner, DL; Dunham, ME; et al. (1998 ) Ammonia capsule ingestion causing upper aerodiges tive tract injury. Otolaryngol Head Neck Surg 119(6):6 78–680.

Rosenfeld, M. (1932) Experimental modification of m itosis by ammonia. Arch Exp Zellforsch Besonders Gewebezuecht 14:1–13. (German)

Rosswall, T. (1981) The biogeochemical nitrogen cyc le. In: Likens, GE, ed. Some perspectives of the m ajor biogeochemical cycles. Chichester, England: John Wi ley & Sons, pp. 25–49.

Sabuncuoglu, N; Coban, O; Lacin, E; et al. (2008) E ffect of barn ventilation on blood gas status and s ome physiological traits of dairy cows. J Environ Biol 29(1):107–110.

Sadasivudu, B; Murthy, RK. (1978) Effects of ammoni a on monoamine oxidase and enzymes of GABA metaboli sm in mouse brain. Arch Int Physiol Biochim 86:67–82.

Sadasivudu, B; Rao, TI; Radhakrishna, C. (1977) Acu te metabolic effects of ammonia in mouse brain. Ne urochem Res 2:639–655.

Sadasivudu, B; Rao, TI; Radhakrishna, M. (1979) Chr onic metabolic effects of ammonia in mouse brain. Arch Int Physiol Biochim 87:871–885.

Sadasivudu, B; Murthy, CRK; Rao, GN; et al. (1983) Studies on acetylcholinesterase and gammaglutamyltranspeptides in mouse brain in ammonia tox icity. J Neurosci Res 9:127–134.

Sanchez-Perez, AM; Felipo, V. (2006) Chronic exposu re to ammonia alters basal and NMDA-induced phosphorylation of NMDA receptor-subunit NR1. Neur oscience 140(4):1239–1244.

Schaerdel, AD; White, WJ; Lang, CM; et al. (1983) L ocalized and systemic effects of environmental ammo nia in rats. Lab Anim Sci 33(1):40–45.

Schoeb, TR; Davidson, MK; Lindsey, JR. (1982) Intra cage ammonia promotes growth of micoplasma pulmonis in the respiratory tract of rats. Infect Immun 38:212 –217.

Schubiger, G; Bachmann, C; Barben, P; et al. (1991) N-acetylglutamate synthetase deficiency: diagnosis

, management and follow-up of a rare disorder of ammo nia detoxication. Eur J Pediatr 150(5):353–356.

### 80 DRAFT - DO NOT CITE OR QUOTE

Shawcross, D; Jalan, R. (2005) The pathophysiologic basis of hepatic encephalopathy: central role for ammonia and inflammation. Cell Mol Life Sci 62(19-20):2295–230 4.

Shawcross, DL; Wright, G; Olde Damink, SW; et al. ( 2007) Role of ammonia and inflammation in minimal h epatic encephalopathy. Metab Brain Dis 22(1):125–138.

Shimizu, H; Suzuki, Y; Takemura, N; et al. (1985) R esults of microbial mutation test for forty-three i ndustrial chemicals. Sangyo Igaku 27(6):400–419.

Sigurdarson, ST; O'Shaughnessy, PT; Watt, JA; et al . (2004) Experimental human exposure to inhaled gra in dust and ammonia: towards a model of concentrated animal feeding operations. Am J Ind Med 46(4):345–348.

Silver, SD; McGrath, FP. (1948) A comparison of acu te toxicities of ethylene imine and ammonia to mice . J Ind Hyg Toxicol 30:7–9.

Silverman, L; Whittenberger, JL; Muller, J. (1949) Physiological response of man to ammonia in low concentrations. J Ind Hyg Toxicol 31:74–78.

Sjoblom, E; Hojer, J; Kulling, PE; et al. (1999) A placebo-controlled experimental study of steroid in halation therapy in ammonia-induced lung injury. J Toxicol Clin Toxicol 37(1):59–67.

Skowronska, M; Zielinska, M; Albrecht, J. (2009) Na triuretic peptides differently modulate accumulatio n of reactive oxygen species and nitric oxide in cultured astrocy tes: effect of treatment with ammonia. J Neurochem 110(Suppl-1):7.

Slot, GMJ. (1938) Ammonia gas burns. Lancet (Dec 1 0):1356–1357.

Smeets, MAM; Bulsing, PJ; van Rooden, S; et al. (20 07) Odor and irritation thresholds for ammonia: a c omparison between static and dynamic olfactometry. Chem Sens es 32(1):11–20.

Smith, D; Spanel, P; Davies, S. (1999) Trace gases in breath of healthy volunteers when fasting and after a proteincalorie meal: a preliminary study. J Appl Physiol 87(5):1584–1588.

Smith, D; Wang, T; Pysanenko, A; et al. (2008) A se lected ion flow tube mass spectrometry study of amm onia in mouth- and nose-exhaled breath and in the oral cavi ty. Rapid Commun Mass Spectrom 22(6):783–789.

Sobonya, R. (1977) Fatal anhydrous ammonia inhalati on. Hum Pathol 8:293–299.

Socolow, RH. (1999) Nitrogen management and the fut ure of food: Lessons from the management of energy and carbon. Proc Natl Acad Sci (USA) 6:6001–6008.

Sorensen, M; Munk, OL; Keiding, S. (2009) Backflux of ammonia from brain to blood in human subjects wi th and without hepatic encephalopathy. Metab Brain Dis 24 (1):237–242.

Sotiropoulos, G; Kilaghbian, T; Dougherty, W; et al . (1998) Cold injury from pressurized liquid ammoni a: a report of two cases. J Emerg Med 16(3):409–412.

Souba, WW. (1987) Interorgan ammonia metabolism in health and disease: a surgeon's view. J Parenter E nteral Nutr 11(6):569–579.

Spanel, P; Dryahina, K; Smith, D. (2007a) The conce ntration distributions of some metabolites in the e xhaled breath of young adults. J Breath Res 1(2):026001. Availab le online at http://iopscience.iop.org/1752- 7163/1/2/026001/pdf/1752-7163_1_2_026001.pdf (acces sed December 22, 2010).

Spanel, P; Dryahina, K; Smith, D. (2007b) Acetone, ammonia and hydrogen cyanide in exhaled breath of s everal volunteers aged 4–83 years. J Breath Res 1(1):0110 01 Available online at http://iopscience.iop.org/17 52- 7163/1/1/011001/pdf/1752-7163_1_1_011001.pdf (acces sed December 22, 2010).

### 81 DRAFT - DO NOT CITE OR QUOTE

Stabenau, JR; Warren, KS; Rall, DP. (1958) The role of pH gradient in the distribution of ammonia betw een blood and cerebro-spinal fluid, brain and muscle. J Clin Invest 38:373–383.

Stombaugh, DP; Teague, HS; Roller, WL. (1969) Effec ts of atmospheric ammonia on the pig. J Anim Sci 2 8:844– 847.

Stroud, S. (1981) Ammonia inhalation: a case report . Crit Care Nurse 1:23–26.

Summerskill, WHJ; Wolpert, E. (1970) Ammonia metabo lism in the gut. Am J Clin Nutr 23:633–639.

Sundblad, BM; Larsson, BM; Acevedo, F; et al. (2004 ) Acute respiratory effects of exposure to ammonia on healthy persons. Scand J Work Environ Health 30(4):313–321

.

Suzuki, H; Yanaka, A; Nakahara, A; et al. (2000) Th e mechanism of gastric mucosal apoptosis induced by ammonia. Gastroenterology 118(Suppl 2):A734.

Swamy, M; Zakaria, AZ; Govindasamy, C; et al. (2005 ) Effects of acute ammonia toxicity on nitric oxide (NO), citrulline-NO cycle enzymes, arginase and related m etabolites in different regions of rat brain. Neur osci Res 53(2):116–122.

Takagaki, G; Berl, S; Clarke, DD; et al. (1961) Glu tamic acid metabolism in brain and liver during inf usion with ammonia labelled with nitrogen-15. Nature 189:326.

Takeuchi, K; Ohuchi, T; Harada, H; et al. (1995) Ir ritant and protective action of urea-urease ammonia in rat gastric mucosa. Dig Dis Sci 40(2):274–281.

Taplin, GV; Chopra, S; Yanda, RL; et al. (1976) Rad ionucliaic lung-imaging procedures in the assessmen t of injury due to ammonia inhalation. Chest 69:582–586.

Targowski, SP; Klucinski, W; Babiker, S; et al. (19 84) Effect of ammonia on in vivo and in vitro immun e response. Infect Immun 43(1):289–293.

Tepper, A; Constock, GW; Levine, M. (1991) A longit udinal study of pulmonary function in fire fighters . Am J Ind Med 20(3):307–316.

Tonelli, AR; Pham, A. (2009) Bronchiectasis a long- term sequela of ammonia inhalation: a case report a nd review of the literature. Burns 35(3):451–453.

Tong, XY; Bethea, JR; Jayakumar, AR; et al. (2009) Role of NF- κ B in ammonia-induced astrocyte swelling and brain edema in acute liver failure. J Neurochem 10 8(Suppl. 1):68.

Toth, B. (1972) Hydrazine, methylhydrazine and meth ylhydrazine sulfate carcinogenesis in Swiss mice. F ailure of ammonium hydroxide to interfere in the development of tumors. Int J Cancer 9:109–118.

Tsujii, M; Kawano, S; Tsuji, S; et al. (1992a) A po ssible promotor in Helicobacter pylori -related gastric carcinogenesis. Cancer Lett 65(1):15–18.

Tsujii, M; Kawano, S; Tsuji, S; et al. (1992b) Mech anism of gastric mucosal damage induced by ammonia. Gastroenterology 102:1881-1888.

Tsujii, M; Kawano, S; Tsujii, S; et al. (1993) Cell kinetics of mucosal atrophy in rat stomach induced by long-term administration of ammonia. Gastroenterology 104(3) :796–801.

Tsujii, M; Kawano, S; Tsujii, S; et al. (1995) Mech anism for ammonia-induced promotion of gastric carc inogenesis in rats. Carcinogenesis 16(3):563–566.

Turner, C; Spanel, P; Smith, D. (2006) A longitudin al study of ammonia, acetone and propanol in the ex haled breath of 30 subjects using selected ion flow tube mass sp ectrometry, SIFT-MS. Physiol Meas 27(4):321–337.

### 82 DRAFT - DO NOT CITE OR QUOTE

Urbain, B; Gustin, P; Prouvost, JF; et al. (1994) Q uantitative assessment of aerial ammonia toxicity t o the nasal mucosa by use of the nasal lavage method in pigs. Am J Vet Res 55(9):1335–1340.

U.S. EPA (Environmental Protection Agency). (1986a) Guidelines for the health risk assessment of chemi cal mixtures. Federal Register 51(185):34014–34025. Ava ilable online at http://www.epa.gov/iris/backgrd.ht ml.

U.S. EPA (Environmental Protection Agency). (1986b) Guidelines for mutagenicity risk assessment. Feder al Register 51(185):34006–34012. Available online at h ttp://www.epa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (1988) Recommendations for and documentation of biological values for use in risk assessment. Prepared by the Enviro nmental Criteria and Assessment Office, Office of H ealth and Environmental Assessment, Cincinnati, OH for the Of fice of Solid Waste and Emergency Response, Washing ton, DC; EPA 600/6-87/008. Available online at http://ww w.epa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (1991) Guidelines for developmental toxicity risk assessme nt. Federal Register 56(234):63798–63826. Available onl ine at http://www.epa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (1994a) Interim policy for particle size and limit concent ration issues in inhalation toxicity studies. Federal Register 5 9(206):53799. Available online at http://www.epa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (1994b) Methods for derivation of inhalation reference concentrations and application of inhalation dosime try. Office of Research and Development, Washingto n, DC; EPA/600/8-90/066F. Available online at http://www.e pa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (1995) Use of the benchmark dose approach in health risk assessment. Risk Assessment Forum, Washington, DC; EPA/630/R-94/007. Available online at http://www.epa.gov/raf/publications/pdfs/BENCHMARK. PDF.

U.S. EPA (Environmental Protection Agency). (1996) Guidelines for reproductive toxicity risk assessmen t. Federal Register 61(212):56274–56322. Available online at h ttp://www.epa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (1998) Guidelines for neurotoxicity risk assessment. Fede ral Register 63(93):26926–26954. Available online at ht tp://www.epa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (2000a) Science policy council handbook: risk characteriza tion. Office of Science Policy, Office of Research and De velopment, Washington, DC; EPA 100-B-00-002. Availa ble online at http://www.epa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (2000b) Benchmark dose technical guidance document. Exter nal review draft. Risk Assessment Forum, Washington, DC ; EPA/630/R-00/001. Available online at http://www.epa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (2000c) Supplementary guidance for conducting health risk assessment of chemical mixtures. Risk Assessment F orum, Washington, DC; EPA/630/R-00/002. Available o nline at http://www.epa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (2002) A review of the reference dose and reference concen tration processes. Risk Assessment Forum, Washington, DC; EPA/630/P-02/0002F. Available online at http://www.epa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (2005a) Guidelines for carcinogen risk assessment. Risk A ssessment Forum, Washington, DC; EPA/630/P-03/001F. Available online at http://www.epa.gov/iris/backgrd.html.

U.S. EPA (Environmental Protection Agency). (2005b) Supplemental guidance for assessing susceptibility from early-life exposure to carcinogens. Risk Assessmen t Forum, Washington, DC; EPA/630/R-03/003F. Availab le online at http://www.epa.gov/iris/backgrd.html.

### 83 DRAFT - DO NOT CITE OR QUOTE

U.S. EPA (Environmental Protection Agency). (2006a) Science policy council handbook: peer review. Thi rd edition. Office of Science Policy, Office of Resear ch and Development, Washington, DC; EPA/100/B-06/00 2. Available online at http://www.epa.gov/iris/backgrd .html.

U.S. EPA (Environmental Protection Agency). (2006b) A framework for assessing health risk of environme ntal exposures to children. National Center for Environ mental Assessment, Washington, DC, EPA/600/R-05/093 F. Available online at http://cfpub.epa.gov/ncea/cfm/r ecordisplay.cfm?deid=158363.

U.S. EPA (Environmental Protection Agency). (2011a) Recommended use of body weight 3/4

as the default method in derivation of the oral ref erence dose. Risk Assessment Forum, Washington, DC; EPA/100/R11/0001. http://www.epa.gov/raf/publicatio ns/interspecies-extrapolation.htm.

U.S. EPA (Environmental Protection Agency). (2011b) Exposure factors handbook 2011 edition (final). Na tional Center for Environmental Assessment, Washington, DC , EPA/600/R-09/052F. Available online at http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid =236252

Uzvolgyi, E; Bojan, F. (1980) Possible in vivo form ation of a carcinogenic substance from diethyl pyro carbonate and ammonia. J Cancer Res Clin Oncol 97:205–207.

van de Poll, MC; Ligthart-Melis, GC; Olde Damink, S W; et al. (2008) The gut does not contribute to sys temic ammonia release in humans without portosystemic shu nting. Am J Physiol Gastrointest Liver Physiol 295(4):G760–G765.

Van Slyke, DD; Phillips, RA; Hamilton, PB; et al. ( 1943) Glutamine as source material of urinary ammon ia. J Biol Chem 150:481–-482.

Verberk, MM. (1977) Effects of ammonia in volunteer s. Int Arch Occup Environ Health 39:73–81.

Verschueren, K. (2001) Ammonia. In: Handbook of en vironmental data on organic chemicals. New York, NY : John Wiley & Sons, Inc., pp. 21–24, 188–189.

Vogelzang, PFJ; van der Gulden, JWJ; Preller, L; et al. (1997) Bronchial hyperresponsiveness and expos ure in pig farmers. Int Arch Occup Environ Health 70(5):327–3 33.

Vogelzang, PFJ; van der Gulden, JWJ; Folgering, H; et al. (1998) Longitudinal changes in lung function associated with aspects of swine-confinement exposure. J Occu p Environ Med 40:1048–1052.

Vogelzang, PFJ; van der Gulden, JWJ; Folgering, H; et al. (2000) Longitudinal changes in bronchial res ponsiveness associated with swine confinement dust exposure. C hest 117(5):1488–1495.

Walton, M. (1973) Industrial ammonia gassing. Br J Ind Med 30:78–86.

Wands, RC (1981) Alkaline materials. In: Clayton, FE and Clayton, FE; eds. Patty's industrial hygiene and toxicology. 4th edition. New York, NY: John Wiley & Sons, Inc., pp. 3045–3069.

Ward, K; Costello, GP; Murray, B. (1983) Acute and long-term pulmonary sequelae of acute ammonia inhal ation. Irish Med J 76(6):279–281.

Warren, KS. (1958) The differential toxicity of amm onia salts. J Clin Invest 37:497–501.

Wason, S; Stepman, M; Breide, C. (1990) Ingestion o f aromatic ammonia smelling salts capsules. Am J D is Child 144(2):139–140.

Weatherby, JH. (1952) Chronic toxicity of ammonia f umes by inhalation. Proc Soc Exp Biol Med 81:300–3 01.

Welch, A. (2006) Exposing the dangers of anhydrous ammonia. Nurse Pract 31(11):40–45.

Weiser, JR; Mackenroth, T. (1989) Acute inhalatory mass ammonia intoxication with fatal course. Exp P athol 37(1–4):291–295.

### 84 DRAFT - DO NOT CITE OR QUOTE

White, CE; Park, MS; Renz, EM; et al. (2007) Burn c enter treatment of patients with severe anhydrous a mmonia injury: case reports and literature review. J Burn Care Res 28(6):922–928.

White, ES. (1971) A case of near fatal ammonia gas poisoning. J Occup Med 13:543–550.

Whittaker, AG; Love, S; Parkin, TD; et al. (2009) S tabling causes a significant increase in the pH of the equine airway. Equine Vet J 41(9):940–943.

WHO (World Health Organization). (1986) Ammonia. En vironmental Health Criteria 54. Available online a t http://www.inchem.org/documents/ehc/ehc/ehc54.htm ( accessed August 18, 2009).

Xue, Z; Li, B; Gu, L; et al. (2010) Increased Na, K -ATPase α 2 isoform gene expression by ammonia in astrocytes and in brain in vivo. Neurochem Int 57(4):395–403.

Yadav, JS; Kaushik, VK. (1997) Genotoxic effect of ammonia exposure on workers in a fertility factory. Indian J Exp Biol 35(5):487–492.

Yang, GY. (1987) An industrial mass ammonia exposur e. Vet Hum Toxicol 29:476–477.

Zander, DL; Thompson, JG; Lane, M. (2006) Ammonium impairs mitochondrial function and homeostasis in Murine 2-cell embryos. Biol Reprod (Spec Iss):125– 126.

Zejda, JE. (1994) Respiratory health status in swin e producers relates to endotoxin exposure in the pr esence of low dust levels. J Occup Med 36(1):49–56.

Zielinska, M; Law, RO; Albrecht, J. (2003) Excitoto xic mechanism of cell swelling in rat cerebral cort ical slices treated acutely with ammonia. Neurochem Int 43(4–5 ):299–303.

### A-1 DRAFT - DO NOT CITE OR QUOTE

APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PU BLIC 1 

### COMMENTS AND DISPOSITION 2

3

4

5

[To be added] 6 7B-1 DRAFT - DO NOT CITE OR QUOTEAPPENDIX B. SUMMARY OF REPEAT DOSE TOXICITY INFORMATION FOR SELECTED AMMONIUM SALTS123Table B-1. Summary of noncancer results of repeatdose studies of oral exposure of experimental animals to selectedammonium saltsStrain/species/sexAmmoniaspeciesDose(mg/kg-d)/durationNOAEL(mg/kg-d)LOAEL(mg/kg-d)Effects at the LOAELdose Comment ReferenceSubchronic studiesWistar rat(10/sex/group)Ammoniumchloride0, 1,590, or3,050 (males);0, 1,800, or3,700 (females)for 13 wks(administeredin diet)Not determined1,590 (males)1,800 (females)Decreased body weights (6-17% in males; 11-19% infemales), changes in serumchemistry (increased plasmachloride and ALP activity),increased relative kidneyweights (both dose levels,7-28%) and adrenal weights(high-dose males, 18%),metabolic acidosis (malesand females) andsubsequent hypertrophy ofthe adrenal zonaglomerulosa (males only)Lina and Kuijpers,2004Wistar rat(5 females/group)Ammoniumacetate in dietand drinkingwater17.1 or 2,370 indiet; and 0 or42.8 in watercontinuouslyfor 90 dNot determined2,410 (combineddose)Depression in body weightgainControl diet contained 0.024%ammonia. Authors suggestedthat the transient effects onGFAP protein and comparableblood levels were indicativeof an adaptive response.Bodega et al., 1993B-2 DRAFT - DO NOT CITE OR QUOTETable B-1. Summary of noncancer results of repeatdose studies of oral exposure of experimental animals to selectedammonium saltsStrain/species/sexAmmoniaspeciesDose(mg/kg-d)/durationNOAEL(mg/kg-d)LOAEL(mg/kg-d)Effects at the LOAELdose Comment ReferenceChronic studiesSprague-Dawleyrat(11 males/group)Ammoniumchloride0 or 1,800 for330 d(administeredin drinkingwater)Not determined1,800Depression in body weights(13-20% with regular andlow-calcium diets,respectively), metabolicacidosis, and loss of bone(femur) tissueMetabolic acidosis (reducedblood pH and plasma carbondioxide).Combined effects of calciumintake and observed that theloss of bone tissue wasindependent of the level ofdietary calcium.Barzel et al., 1969Wistar rat(15/sex/group)Ammoniumchloride0, 481, or1,020 (males);0, 610, or 1,370(females) for18 moNot determined481 (males)610 (females)Metabolic acidosis Femur weight significantlyincreased (~17%) in high-dosemales. Adrenal and kidneyweights elevated (<15%).Increased incidence of zonaglomerulosa hypertrophy ofthe adrenal at high dose (notstatistically significant).Severity of metabolic acidosisincreased with dose.Lina and Kuijpers,2004Wistar rat(50/sex/group)Ammoniumchloride0, 455, or1,000 (males);0, 551, or 1,200(females) for30 moNot determined455 (males)551 (females)Metabolic acidosis and anincreased incidence ofhypertrophy of the adrenalglomerulosa (males only)Increased incidence of zonaglomerulosa hypertrophy ofthe adrenal in high-dosefemales; attributed to chronicstimulation of the adrenalcortex by ammonium chlorideinduced acidosis. There wasno evidence of a carcinogenicresponse.Lina and Kuijpers,2004B-3 DRAFT - DO NOT CITE OR QUOTETable B-1. Summary of noncancer results of repeatdose studies of oral exposure of experimental animals to selectedammonium saltsStrain/species/sexAmmoniaspeciesDose(mg/kg-d)/durationNOAEL(mg/kg-d)LOAEL(mg/kg-d)Effects at the LOAELdose Comment ReferenceF344 rat(10/sex/group)Ammoniumsulfate0, 42, 256, or1,527 (males);0, 48, 284, or1,490 (females)for 52 wks(administeredin diet)256 (males)284 (females)1,527 (males)1,490 (females)Elevated relative liver andkidney weights (7-10%)No significant effects onhematology, serum chemistry,or histopathology.Ota et al., 2006F344 rat(50/sex/group)Ammoniumsulfate0, 564, or 1,288(males); 0, 650,or 1,371(females) for104 wks(administeredin diet)1,288 (males)1,371 (females)Not determinedClinical chemistry andhematology not evaluated;organ weights not measured.Incidence of chronicnephropathy in male ratsincreased over control (1/48,5/49, 3/48 in the control, midand high dose); increase wasstatistically significant only atthe mid-dose and not doserelated. There was noevidence of a carcinogenicresponse.Ota et al., 2006Developmental studiesWistar rat(group sizes ofpregnant rats notreported)Ammoniumacetate0 or21,000 startingon d 1 ofpregnancythroughweaning toPND 21Not determined(maternal ordevelopmental)Maternal effectsnot reported21,000(developmental)Reduction in pup bodyweight gainPups exposed to ammoniaduring gestation and lactationonly were resistant to acuteammonia toxicity induced bya single injection of 540mg/kg ammonium acetate.Minana et al., 1995

### C-1 DRAFT - DO NOT CITE OR QUOTE APPENDIX C. SUPPLEMENTAL INFORMATION ON AMMONIA 1

2



### C.1. AMMONIA LEVELS MEASURED IN EXPIRED AIR IN HUM ANS 3 

### Available data on ammonia levels in the expired air of volunteers are summaried in 4



### Table C-1. 5C-2 DRAFT - DO NOT CITE OR QUOTETable C-1. Ammonia levels in exhaled breath of volunteersBreath samples from the nose and tracheaTest subjects Breath samplesLevels of ammonia in exhaledbreath MethodsComments ReferenceThree healthy malevolunteers (>30 yrs ofage)Ammonia levels measured in noseexhaled breath of test subjects eachmorning about 2 hrs after eating aregular breakfast; samplescollected daily over a 4-wk periodVolunteer A = 103 ± 1.2 ppb (0.0728± 0.000848 mg/m3)Volunteer B = 110 ±1.3 ppb (0.0777 ±0.000919 mg/m3)Volunteer C = 83 ± 1.2 ppb (0.0587 ±0.000848 mg/m3)(median ammonia levels estimated asgeometric mean ± geometric standarddeviation)SIFT-MS analysisMean ambient air level ofammonia was 80 ± 10 ppb(0.056 ± 0.0071 mg/m3)The authors indicated thatammonia measured inmouth-exhaled breath maybe generated in the oralcavity and suggested thatconcentrations in noseexhaled breath may betterrepresent systemicconditions (such asmetabolic disease)Smith et al.,2008Sixteen healthy subjects(9 males aged 25–63 yrsand 7 females aged 23–41 yrs); subgroups testedwere all maleBreath samples collected duringquiet nose breathing, and directsampling during a deep inspirationfollowed by breath-holding withthe glottis closedAmmonia concentrations ranged from0.013 to 0.046 mg/m3during nosebreathing (median 0.025 mg/m3) (5male subjects), and 0.029 mg/m3froman air sampled collected from thetrachea (collected from a tube insertedinto one male subject’s nose and intothe trachea)Chemiluminescence Larson et al.,19771C-3 DRAFT - DO NOT CITE OR QUOTETable C-1. Ammonia levels in exhaled breath of volunteersBreath samples from the mouth and oral cavityTest subjects Breath samplesLevels of ammonia in exhaledbreath MethodsComments ReferenceThree healthy malevolunteers (>30 yrs ofage)Ammonia levels measured inmouth-exhaled breath and in theclosed mouth cavity of testsubjects each morning about 2 hrsafter eating a regular breakfast;samples collected daily over a 4-wk periodvia Mouth:Volunteer A = 1,088 ± 1.3 ppb (0.769± 0.000919 mg/m3)Volunteer B = 885 ± 1.3 ppb (0.626 ±0.000919 mg/m3)Volunteer C = 855 ± 1.3 ppb (0.604 ±0.000919 mg/m3)via Oral Cavity:Volunteer A = 1,465 ± 1.4 ppb (1.04 ±0.000990 mg/m3)Volunteer B = 2,146 ± 1.5 ppb (1.52 ±0.00106 mg/m3)Volunteer C = 1,859 ± 1.3 ppb (1.31 ±0.000919 mg/m3)(median ammonia levels estimated asgeometric mean ± geometric standarddeviation)SIFT-MS analysisMean ambient air level ofammonia was 80 ± 10 ppb(0.056 ± 0.0071 mg/m3)The authors indicated thatammonia measured inmouth-exhaled breath maybe generated in the oralcavity and suggested thatconcentrations in noseexhaled breath may betterrepresent systemicconditions (such asmetabolic disease)Smith et al.,2008Twenty-six secondaryschool students (10 malesand 16 females, 17–18 yrsold and one 19 yr old)Three sequential breath exhalationscollected over 5 min following thestudents listening to a 1-hrpresentation (at least 1 hrfollowing breakfast and beforelunch); alveolar portion measured(identified using humidity)Median values reported for:17 yr olds = 233 ppb (0.165 mg/m3)18 yr olds = 346 ppb (0.245 mg/m3)SIFT-MS analysisSignificant differences inammonia levels in exhaledbreath between 17 and18 yr olds (p< 10-8) werereportedSpanel et al.,2007aC-4 DRAFT - DO NOT CITE OR QUOTETable C-1. Ammonia levels in exhaled breath of volunteersBreath samples from the mouth and oral cavityTest subjects Breath samplesLevels of ammonia in exhaledbreath MethodsComments ReferenceFour healthy children(2 males and 2 females,4–6 yrs old)Thirteen senior volunteers(11 males and 2 females,60–83 yrs old); four hadtype-2 diabetes mellituswith onset at agesbetween 50 and 70 yrs,and controlled by dietAll subjects had theirregular breakfast withoutany specific restrictionsBreath samples collected inmorning at least 1 hr afterbreakfast and at least 1 hr prior tolunch; each volunteer performedtwo exhalation/inhalation cycles(both about 5–10 sec in duration)Children = range 223–643 ppb(0.157–0.454 mg/m3)Seniors = 317–2,091 ppb (0.224–1.48 mg/m3)SIFT-MS analysisAmmonia breath levelssignificantly increased withageSome seniors reporteddiabetesMeasured ammonia level inbreath reported for eachsubjectSpanel et al.,2007bThirty healthy volunteers(19males and 11 females,24–59 yrs, 28 Caucasian,1 African, and 1 mixedrace); volunteers wereinstructed to maintaintheir normal dailyroutines and to not rinseout their mouths prior toproviding a breath sampleBreath samples collected in themorning prior to lunch atapproximately weekly intervals forabout 6 mo; some volunteersprovided samples more frequentlythan others; 480 samples collectedand analyzed for ammoniaGeometric mean and geometricstandard deviation = 833 ± 1.62 ppb(0.589 ± 0.00114 mg/m3)Median = 842 ppb (0.595 mg/m3)Range = 248–2,935 ppb (0.175–2.08 mg/m3)SIFT-MS analysisAmmonia breath levelswere shown to increasewith ageBackground levels in thetesting laboratory weretypically around 400 ppb(0.28 mg/m3)Turner et al.,2006Five subjects (2 females,3 males; age range 27–65 yrs)Breath samples collected between8 and 9 am in three sequentialbreath exhalations on multiple days(12–30 d) over the course of amonthAmmonia concentrations ranged from422 to 2,389 ppb (0.298–1.69 mg/m3)SIFT-MS analysisDifferences in ammoniabreath levels betweenindividuals were significant(p< 0.001)Diskin et al.,2003C-5 DRAFT - DO NOT CITE OR QUOTETable C-1. Ammonia levels in exhaled breath of volunteersBreath samples from the mouth and oral cavityTest subjects Breath samplesLevels of ammonia in exhaledbreath MethodsComments ReferenceSix normal nonsmokingmale volunteers (24–61 yrs old), fasted for12 hrs prior to testingBaseline breath sample obtained;breath samples collected 20, 40,and 60 min and 5 hrs following theingestion of a liquid protein-caloriemealPremeal levels ranged from 300 to600 ppb (0.2–0.4 mg/m3);Postmeal levels at 30 min were200 ppb (0.1 mg/m3) increasing tomaximum values at 5 hrs of 600–1,800 ppb (0.4–1.3 mg/m3)SIFT-MS analysis A biphasic response inbreath ammoniaconcentration was observedafter eatingSmith et al.,1999Fourteen healthy,nonsmoking subjects (agerange 21–54 yrs)performed one or more ofthe following hygienemaneuvers:(1) acidic oral rinse(pH 2.5),(2) tooth brushingfollowed by acidic oralrinse,(3) tooth brushingfollowed by distilledwater rinse, and(4) distilled water rinseSubjects fasted for 8 hrs prior tobaseline measurement, refrainedfrom oral hygiene after their mostrecent meal, refrained from heavyexercise for 12 hrs, and had noliquid intake for several hours;initial breath ammonia wasmeasured between 8 and 10 am,then subjects performed one ormore of the hygiene measureslisted (at 30-min intervals for atotal 90-min period; samplescollected over 5 min)Baseline levels varied from 120 to1,280 ppb (0.085–0.905 mg/m3)Nitrogen oxideanalyzer with anammonia conversionchannel (similar tochemiluminescence)An 80–90% depletion ofvolatile ammonia emissionswas seen within 10 min ofacid rinsing; less than a50% depletion of ammoniawas seen following toothbrushing or distilled waterrinse; gaseous ammonialevels increased after allrinse procedures over timeNorwood etal., 1992Sixteen healthy subjects(9 males aged 25–63 yrsand 7 females aged 23–41 yrs); subgroups testedwere all maleBreath samples collected duringquiet mouth breathingAmmonia concentrations ranged from0.029 to 0.52 mg/m3during mouthbreathing (median of 0.17 mg/m3)Chemiluminescence The oral cavity appears tobe a source of breathammonia; no attempt wasmade to control the diet ofsubjects or standardize theinterval between the lastmeal and the measurementLarson et al.,19771C-6 DRAFT - DO NOT CITE OR QUOTETable C-1. Ammonia levels in exhaled breath of volunteersBreath samples: source (nose/mouth/oral cavity) notspecifiedTest subjects Breath samplesLevels of ammonia in exhaledbreath MethodsComments ReferenceSixteen healthy,nonsmoking subjects(4 females and 12 males,29 ± 7 yrs); no significantdifferences in mean age,height, weight, body massindex (BMI), or timesince last oral intake;10 subjects tested in eachexperimentExperiment 1: single whole-breathsamples collected from eachsubject (same samples immediatelyreanalyzed within <10 sec to assessinstrument specific variability)Experiment 2: three repeat breathsamples collected from eachsubject (to evaluate intra-subjectdifferences); this experimentevaluated differences based onstandardization of expiratorypressure and flowExperiment 3: two mixed breathsamples and two bag alveolarbreath samples collected in shortsuccession from each subjectExperiment 1: 1,192 ± 85 ppb(0.843 ± 0.0601 mg/m3;median ± measurement error)Experiment 2:Nonstandardized = 1,007 ± 184 ppb(0.712 ± 0.130 mg/m3; median ±standard deviation)Standardized = 1,433 ± 160 ppb(1.01 ± 0.113 mg/m3; median ±standard deviation)Experiment 3:Mixed = 1,216 ± 827 ppb (0.860 ±0.585 mg/m3; median ± standarddeviation)Alveolar = 1,301 ± 791 ppb (0.920 ±0.559 mg/m3; median ± standarddeviation)SIFT-MS analysisThis study establishesthat SIFT-MS analysisis reliable andrepeatableRelatively small number ofhealthy subjects usedDoes not address the breathof those with diseaseIntra-day and inter-dayrepeatability were notinvestigatedBoshier et al.,2010C-7 DRAFT - DO NOT CITE OR QUOTETable C-1. Ammonia levels in exhaled breath of volunteersBreath samples: source (nose/mouth/oral cavity) notspecifiedTest subjects Breath samplesLevels of ammonia in exhaledbreath MethodsComments ReferenceEight healthy subjects(average age 39.8 ±9.6 yrs)Subjects fasted for 6 hrs prior tosamples being collected. Subjectsbreathed normally into collectiondevice for 5 minMean breath ammonia = 0.49 ±0.24 ppm (0.35 ± 0.17 mg/m3)Fiber optic sensor This study measuredammonia levels in healthyvolunteers compared toHelicobacter pyloripositive individuals (fivesubjects) (data not shown);the experiment alsoincluded a challenge with a300 mg urea capsule toevaluate the urease activityof healthy vs. infectedindividuals (data notshown); the authorsconcluded that breathammonia measurement maybe feasible as a diagnostictest forH. pyloriKearney et al.,2002Three groups of childrenwere used as test subjects:(1) 68 asthmatic childrenresiding in a NationalPark in the mountains(mean age 10 yrs,48 boys, 20 girls),(2) 52 asthmatic childrenin an urban area (meanage 9 yrs, 35 boys,17 girls), and(3) 20 healthy childrenfrom the same urban areaas a control group (meanage 10, 12 boys, 8 girls)Subjects performed a 5-sec breathhold and exhaled slowly intocollection deviceAsthmatic children from NationalPark = 5.6 ± 4.7 ppb (0.0040 ±0.0033 mg/m3)Asthmatic urban children:Mean NH3= 14.3 ± 10.2 ppb(0.0101 ± 0.00721 mg/m3)Urban control group:Mean NH3= 14.8 ± 10.3 ppb(0.0105 ± 0.00728 mg/m3)ChemiluminescenceBoth groups of asthmaticchildren had some subjectson glucocorticoids, oftencombined with histamineantagonists and/or b2agonists, while others wereleft untreated; ammoniaconcentrations in exhaledbreath appeared to becorrelated with exposure tourban airGiroux et al.,20021

### C-8 DRAFT - DO NOT CITE OR QUOTE

C.2. HUMAN CASE STUDIES AND REPORTS OF HUMAN EXPOS URE TO 1



### AMMONIA 2 

### Case report findings of injury in adults and childr en due to exposure to ammonia via 3 

### inhalation of vapors, dermal contact, or ingestion of household cleaning solutions or ammonia 4 

### inhalant capsules are presented in Table C-2 below and are organized by exposure route. 5 

### Oral exposure to ammonia most commonly involved ing estion of household cleaning 6 

### solutions or biting into the capsules of ammonia sm elling salts, which are commonly found in 7 

### first aid kits. Young children, generally <4 years old, have been reported as “biting into” or 8 

### ingesting smelling salts capsules. The acute effec ts included drooling, erythematous and 9 

### edematous lips, reddened and blistered tongues, dys phagia, vomiting, and oropharyngeal burns 10 

### (Rosenbaum et al., 1998; Wason et al., 1990; Lopez et al., 1988). Delayed effects were not noted 11 

### in these cases. Gilbert (1988) reported ammonia in toxication characterized by lethargy, 12 

### restlessness, irritability, and confusion in a 37-y ear-old man following surgery. Most other cases 13 

### of ammonia ingestion involved household cleaning so lutions and detergents. Many cases were 14 

### intentional; however, not all were fatal. Klein et al. (1985) described two cases of ingestion of 15 

### approximately 30 mL and “two gulps” of Parson’s sud sy ammonia (ammonia 3.6%; pH 11.5), 16 

### respectively. The first case resulted in a white a nd blistered tongue and pharynx, and esophageal 17 

### burns with friable, boggy mucosa; and in the second case, several small esophageal lesions with 18 

### mild to moderate ulceration and some bleeding were reported. There were no oropharyngeal 19 

### burns in the second case and no delayed complicatio ns in either case. Christesen (1995) reported 20 

### that of 11 cases involving accidental or intentiona l ingestion of ammonia water by adults 21 

### (15 years or older), 2 cases exhibited acute respir atory obstruction, and 1 case developed an 22 

### esophageal stricture 3 months postinjury. In cases involving fatalities, evidence of laryngeal and 23 

### epiglottal edema, erythmatous esophagus with severe corrosive injury, and hemorrhagic 24 

### esophago-gastro-duodeno-enteritis was noted (Klein et al., 1985; Klendshoj and Rejent, 1966). 25 

### Dworkin et al. (2004) reported a case of ingestion of contaminated chicken tenders, prepared and 26 

### served in a school cafeteria, by approximately 157 students and 6 teachers. The onset of acute 27 

### symptoms occurred within an hour of ingestion, and included headache, nausea, vomiting, 28 

### dizziness, diarrhea, and burning mouth. In a case of forced ingestion of an unknown quantity of 29 

### dilute ammonia, a 14-year-old boy presented with di fficulty speaking, ataxic gait, isochoric 30 

### pupils, and evidence of brain edema. There were no burns to the eyes or mouth and no 31 

### indication of gastric pathology. It was only after the patient was able to communicate that 32 

### ammonia was involved that appropriate treatment, fo llowed by a satisfactory outcome, was 33



### achieved. 34 

### Inhalation is the most frequently reported route of exposure and cause of morbidity and 35 

### fatality, and often occurs in conjunction with derm al and ocular exposures. Acute effects from 36

### C-9 DRAFT - DO NOT CITE OR QUOTE

inhalation have been reported to range from mild to severe, with mild symptoms consisting of 1 

### nasal and throat irritation, sometimes with perceiv ed tightness in the throat (Price and Watts, 2 

### 2008; Prudhomme et al., 1998; Weiser and Mackenroth , 1989; Yang, 1987; O’Kane, 1983; Ward 3 

### et al., 1983; Caplin, 1941). Moderate effects are described as moderate to severe pharyngitis, 4 

### tachycardia, frothy, often blood-stained sputum, mo derate dyspnea, rapid, shallow breathing, 5 

### cyanosis, some vomiting, transient bronchospasm, ed ema and some evidence of burns to the lips 6 

### and oral mucosa, and localized to general rhonchi i n the lungs (Weiser and Mackenroth, 1989; 7 

### Yang, 1987; O’Kane, 1983; Ward et al., 1983; Countu rier et al., 1971; Caplin, 1941). Severe 8 

### effects include second- and third-degree burns to t he nasal passages, soft palate, posterior 9 

### pharyngeal wall, and larynx; upper airway obstructi on, loss of consciousness, bronchospasm, 10 

### dyspnea, persistent, productive cough, bilateral di ffuse rales and rhonchi, production of large 11 

### amounts of mucous, pulmonary edema, marked hypoxemi a, local necrosis of the lung, 12 

### deterioration of the whole lung, and fatality. Del ayed effects of acute exposure to high 13 

### concentrations of ammonia include bronchiectasis, b ronchitis, bronchospasm/asthma, dyspnea 14 

### upon exertion and chronic productive cough, bronchi olitis/, severe pulmonary insufficiency, and 15 

### chronic obstructive pulmonary disease (Lalic et al. , 2009; Leduc et al., 1992; Bernstein and 16 

### Bernstein, 1989; Flury et al., 1983; Ward et al., 1 983; Stroud, 1981; Close, 1980; Taplin, et al., 17 

### 1976; Walton, 1973; Kass et al., 1972; Slot, 1938). 18 

### Respiratory effects were also observed following ch ronic occupational exposure to 19 

### ammonia. After 18 months and 1 year on the job, re spectively, both men developed cough, chest 20 

### tightness, and wheezing, typically after 2–6 hours from the beginning of each work day, but not 21 

### on weekends or holidays. In another case, progress ive deterioration of the clinical condition of a 22 

### 68-year-old male was documented for 4 years and dev elopment of diffuse interstitial and severe 23 

### restrictive lung disease was reported following lon g-term repetitive occupational exposure to 24 

### ammonia at or above the odor recognition level (Bra utbar et al., 2003). Lee et al. (1993) report a 25 

### case of a 39-year-old man who developed occupationa l asthma 5 months after beginning a job 26 

### requiring the polishing of silverware. The room in which he worked was poorly ventilated. The 27 

### product used contained ammonia and isopropyl alcoho l and the measured ammonia 28 

### concentration in the breathing zone when using this product was found to be 8–15 ppm (6– 29 

### 11 mg/m 3 

### ). 30 

### Acute dermal exposure to anhydrous (liquid) ammonia and ammonia vapor has resulted 31 

### in caustic burns of varying degrees to the skin and eyes. There are numerous reports of 32 

### exposures from direct contact with anhydrous ammoni a in which first-, second-, and third-degree 33



### burns occurred over as much as 50% of the total bod y surface (Lalic et al., 2009; Pirjavec et al., 34 

### 2009; Arwood et al., 1985). Frostbite injury has a lso been reported in conjunction with exposure 35 

### to sudden decompression of liquefied ammonia, which is typically stored at -33°F (George et al., 36

### C-10 DRAFT - DO NOT CITE OR QUOTE

2000; Sotiropoulos et al., 1998; Arwood et al., 198 5). However, direct contact is not a 1 

### prerequisite for burn injury. Several reports have indicated that burns to the skin occurred with 2 

### exposure to ammonia gas or vapor. Kass et al. (197 2) reported one woman with chemical burns 3 

### to her abdomen, left knee, and forearm and another with burns to the feet when exposed to 4 

### anhydrous ammonia gas released from a derailed trai n in the vicinity. Several victims at or near 5



### the scene of an overturned truck that had been carr ying 8,000 gallons of anhydrous ammonia 6 

### were reported as having second- and third-degree bu rns over exposed portions of the body 7 

### (Burns et al., 1985; Close et al., 1980; Hatton et al., 1979). In a case involving a refrigeration 8 

### leak in a poorly ventilated room, workers located i n an adjacent room reported a “burning skin” 9 

### sensation (de la Hoz et al., 1996) while, in anothe r case involving the sudden release of ammonia 10 

### from a pressure valve in a refrigeration unit, one victim received burns to the leg and genitalia 11 

### (O’Kane, 1983). 12 

### In addition to the skin, the eyes are particularly vulnerable to ammonia burns due to the 13 

### highly water-soluble nature of the chemical and the ready dissociation of ammonium hydroxide 14 

### to release hydroxyl ions. When ammonia or ammonia in solution has been splashed or sprayed 15 

### into the face (accidently or intentionally), immedi ate effects include temporary blindness, 16 

### blepharospasm, conjunctivitis, corneal burns, ulcer ation, edema, chemosis, and loss of corneal 17 

### epithelium (George et al., 2000; Helmers et al., 19 71; Highman, 1969; McGuinness, 1969; Levy 18 

### et al., 1964; Abramovicz, 1924). The long-term eff ects included photophobia, progressive loss 19 

### of sensation, formation of bilateral corneal opacit ies and cataracts, recurrent corneal ulcerations, 20 

### nonreactive pupil, and gradual loss of vision (Yang , 1987; Kass et al., 1972; Helmers et al., 21 

### 1971; Highman, 1969; Osmond and Tallents, 1968; Lev y et al., 1964; Abramovicz, 1924). 22 

### White et al. (2007) reported a case with acute bila teral corneal injury that developed into bilateral 23



### uveitis with stromal vascularization and stromal ha ze and scarring, and pigmented keratic 24 

### precipitates that resulted in legal blindness. An increase in intraocular pressure, resembling 25 

### acute-angle closure glaucoma, was reported by Highm an (1969) following ammonia 26 

### intentionally sprayed into the eyes during robbery attempts. 27

28 

### Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference Oral exposure 57-yr-old male Ingested unknown

quantity of dilute ammonium hydroxide (2.4% ammonia) Hemorrhagic esophagogastro-duodeno-enteritis, death Not applicable Klendshoj and Rejent, 1966

### C-11 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 15-yr-old male Ingestion of about 30 mL

Parson’s sudsy ammonia (ammonia 3.6%; pH 11.5) White and blistered tongue and pharynx, esophageal burns with friable, boggy mucosa; no other complications Not applicable Klein et al., 1985

Middle-aged female Ingested “two gulps”

Parson’s sudsy detergent ammonia Several small esophageal lesions with mild to moderate ulceration and some bleeding; no oropharyngeal burns Not applicable Klein et al., 1985

69-yr-old female Ingested an unknown

quantity of Albertson’s lemon ammonia (ammonia concentration 3%) Lethargy, gurgling respiratory sounds, laryngeal, and epiglottal edema, and a friable, erythmatous esophagus with severe corrosive injury Renal failure, death Klein et al., 1985

Eight young children (ages not given) Biting into an unbroken

capsule of aromatic ammonia inhalant Vomiting, drooling, dysphagia, cough, and oropharyngeal burns Not applicable Lopez et al., 1988

Three children, <4 yrs old Ingestion of aromatic

ammonia “smelling salt” capsules that contained 0.33 mL of a mixture of 18% ammonia and 36% alcohol Pain swallowing, reddened and blistered tongues, drooling, erythema, and swelling of lower lip Not applicable Wason et al., 1990

Eleven adults,
≥ 15 yrs Intentional or accidental

ingestion of ammonia

water Two patients exhibited acute respiratory obstruction, no additional details were provided. One patient developed esophageal stricture 3 mo postinjury Christesen, 1995

3-yr-old female Biting into an ammonia

inhalant capsule Drooling with multiple 1–2 cm white, ulcerativelesions on the midposterior upper surface of the tongue, bilaterally on the buccal mucosa, and on the posterior esophageal wall at the junction of the middle and upper thirds of the esophagus Not applicable Rosenbaum et al., 1998

### C-12 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 2-yr-old female Biting into an ammonia

inhalant capsule Drooling, edema, and erythema on the upper and lower lips with areas of desquamation, superficially ulcerative lesions on the anterior dorsum of the tongue; edema and erythema of the supraglottic structures and upper trachea Not applicable Rosenbaum et al., 1998

One hundred fifty seven students (median age 10 yrs) and six teachers Ingestion of ammoniacontaminated chicken tenders (522–2,468 ppm [369–1,749 mg/m 3 ]) ammonia in uncooked chicken tenders) Stomach ache, headache, nausea, vomiting, dizziness, diarrhea, and mouth burning Not applicable Dworkin et al., 2004

14-yr-old male Ingestion of an unknown

quantity of dilute ammonia Difficulty articulating, ataxic gait, isochoric pupils, cranial CT indicating possible brain edema; hematology, and routine biochemical analysis normal, no burns of the eyes or mouth, and no pathology of the respiratory or gastrointestinal tract Not applicable Dilli et al., 2005

Inhalation-only exposure 39-yr-old male Fumes created during use

of a silver polishing product containing isopropyl alcohol and ammonia (~12 ppm [~8 mg/m 3 ]) in a poorly ventilated basement Cough, breathlessness, and wheezing, rhinitis, and tearing Occupational asthma Lee et al., 1993

68-yr-old male Long-term, repetitive

occupational exposure to anhydrous ammonia at or above odor recognition threshold for 15–20 yrs Examination found persistent pulmonary infiltrates, mainly in upper portion of left chest, diagnosed as diffuse interstitial lung disease Progressive deterioration of clinical condition over a 4-yr period, development of diffuse interstitial lung disease, and severe restrictive lung disease, with pulmonary function tests indicating reduced diffusion capacity at 47% Brautbar et al., 2003

### C-13 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference Inhalation/dermal/ocular exposure Dental patient (age not given) A 10% ammonia solution

spilled into eyes while trying to revive patient from a faint Both eyes red, swollen, with mucopurulent secretions, swollen conjunctiva; the lower left cornea and sclera showed loss of epithelium and the entire left cornea was dull Increase in corneal ulceration with hypopyon and perforation was seen after 8 d; severe conjunctivitis still evident 4 mo after exposure Abramovicz, 1924

21-yr-old female Explosion in an ice

cream factory; ammonia pipe burst Shock, second-degree burns of both feet, the right leg, and small area of the right cheek Residual bronchitis (not further described) Slot, 1938

21-yr-old female Explosion in an ice

cream factory; ammonia pipe burst Shock, conjunctivitis of the right eye, severe tracheitis Not applicable Slot, 1938

23-yr-old female Explosion in an ice

cream factory; ammonia pipe burst Severe shock, persistent, blood-stained vomiting, confusion Not applicable Slot, 1938

21-yr-old female Explosion in an ice

cream factory; ammonia pipe burst Shock, hemicranial headache with nausea Residual bronchitis, “fullness of the head”, and body pains; congestion of both lungs Slot, 1938

36-yr-old female Explosion in an ice

cream factory; ammonia pipe burst Burns and shock; existing bronchitis greatly aggravated Anxiety symptoms, insomnia, painful scar Slot, 1938

25-yr-old female Explosion in an ice

cream factory; ammonia pipe burst Extreme shock, grey pallor, burns to the face, eyes, neck, and both arms, difficulty/inability to swallow, labored breathing Pulse often imperceptible, Cheyne-Stokes respiration, fatality; left lung was congested, edematous, and a small hemorrhage at the base, hemorrhagic bronchitis, desquamation, and small epithelial ulcers of the bronchi Slot, 1938

Nine shelter occupants (ages not given) Ammonia condenser leak

in an air-raid shelter (low exposure group) Eye and mouth irritation, pain on swallowing and hoarseness, suffused conjunctivae, swollen eyelids; lips, mouth, and tongue were erythematous with edema in the back of the throat, and a strong smell of ammonia on the breath Not applicable Caplin, 1941

### C-14 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference Twenty seven shelter occupants (ages not given) Ammonia condenser leak

in an air-raid shelter (moderate exposure group; victims in closer proximity to leak) Cough, blood-stained sputum, hoarseness, chest tightness, increased respiration, hyperaemic conjunctiva, lacrimation; moist sounds in the chest; lips, mouth, tongue, and soft palate showed erythema and edema, with areas of denuded epithelium scattered over the buccal mucosa; three cases showed no improvement and fatality occurred within 36 hrs Nine cases showed initial improvement, with signs of bronchopneumonia appearing on d 2 and 3 postinjury; six recovered while three died within 2 d of onset of secondary infection; autopsy on two males revealed intensely inflamed fauces, pharynx, and larynx; trachea, and bronchi denuded of epithelium or inflamed and filled with purulent exudate; lungs deepl y congested with bronchopneumonia Caplin, 1941

Eleven shelter occupants (ages not given) Ammonia condenser leak

in an air-raid shelter (high exposure group, victims closest to leak source with longest exposure time) Slight cyanosis, intense dyspnea, and persistent cough with foamy sputum; weak and rapid pulse, generalized rales, and rhonchi; seven cases showed no improvement, becoming increasingly cyanotic and dysphonic and died within 48 hrs of

exposure The remaining four cases improved without incident Caplin, 1941

17-yr-old male Struck by a jet of

anhydrous ammonia during crop dusting operations Second-degree burns to the face, marked edema of the eyelids, and unable to open eyes; lips, tongue, and buccal mucosa were hyperemic and burned, swollen uvula (twice normal size), and edematous larynx Permanent loss of vision, with the exception of sustained light perception Levy et al., 1964

17-yr-old male Sprayed with anhydrous

ammonia during crop fertilizing operations First- and second-degree burns to the left arm, anterior chest, face, and neck, burning and edema around the mouth and eyes, conjunctiva appeared inflamed and the mouth was edematous with the uvula swollen to twice the normal size, respiration was labored and rhonchi were present throughout the lungs Chronic persistent cough with a small amount of mucoid sputum, no chronic visual effects Levy et al., 1964

### C-15 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 61-yr-old male Struck in the face with a

blast of anhydrous ammonia while fertilizing crops First-degree burns of the arms and axillae, dyspnea, respiratory distress, red and edematous eyes, unable to see clearly, moderate edema of the mouth and throat with no clear view of the larynx, large amounts of bronchial secretions No chronic impairment of pulmonary function or of vision occurred Levy et al., 1964

28-yr-old male Struck in the face by a

spray of anhydrous ammonia while working on a refrigeration unit First- and second-degree burns to the eyelids, face, anterior chest, ears, forehead, and upper arms, edema and erythema of the pharynx, and inspiratory and expiratory rales could be heard over both lung fields Pulmonary function tests 3 yrs later were unremarkable Levy et al., 1964

Male employee (age not given) Exposure for an

unspecified length of time to ammonia fumes created when a tanker overflowed during a filling operation with 25% ammonia water Vomiting and coughing initially, with some difficulty breathing; after 3-hr delay in seeking medical attention, exhibited red and swollen face, conjunctivitis, red and raw mouth and throat with a swollen tongue, loss of speech, dyspnea, with a weak rhonchi in left lung, cardiac arrest and fatality; autopsy findings included necrosis of the lung, inflammation of the bronchioli; the epithelial layer of the trachea and bronchial tubes were denuded, with an absence of secretions Not applicable Mulder et al., 1967

Male victim at a bank robbery (age not given) Ammonia thrown into

face and forced down throat and up the nose Burns to the face, eyes, and mouth, with severe edema of the nasopharynx and glottis The right eye recovered within 4 d; the left eye showed gross chemosis, corneal staining, nonreactive pupil, intense uveitis with aqueous flare and cells Osmond and Tallents, 1968

### C-16 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 57-yr-old male Assaulted with ammonia

squirted into the eyes during a robbery Decreased visual acuity in the left eye, conjunctivitis, necrosis over the tarsus of the lower lid; the pupil was semi-dilated, oval, and fixed, increased interocular pressure Following initial improvement, the left eye became red, inflamed, ris ing interocular pressure on several occasions, each time responding to surgery and treatment, only to recur after 1 or 2 mo of recovery Highman, 1969

46-yr-old male Assaulted with ammonia

squirted into the eyes during a robbery Conjunctivitis in the right eye, the cornea was hazy with edema Four d following discharge, severe anterior uveitis developed, along with recurrent corneal ulceration and diminished corneal sensation Highman, 1969

Female (age not given) Ammonia was thrown

into eyes in the course of a robbery Two hrs postexposure, loss of corneal epithelium, conjunctivitis, stromal haze, fibrinous material in the anterior chamber, iris atrophy, vertical, oval-shaped pupil, and subcapsular lens opacities Band-shaped corneal degeneration and heterochromia of the iris, but no progression of lens opacities (5 mo following exposure) McGuinness, 1969

40-yr-old male Sprayed on face and

chest during a transfer operation involving anhydrous ammonia Facial burns (not extremely serious), pulmonary edema, and pneumonitis with inflammation and edema of the upper airways No residual lung damage Helmers et al., 1971

17-yr-old farmer Sprayed with several

gallons of 25% ammonia in water during transfer operation Throat tightness during first few minutes following exposure; second-degree burns to the buttocks No residual effects Helmers et al., 1971

36-yr-old male Sprayed in the face

during field repairs to fertilization equipment Immediate blepharospasm, second-degree facial burns, irritative conjunctivitis, superficial corneal ulceration, and palpebral edema of the left eye No known sequelae Helmers et al., 1971

45-yr-old farmer Sprayed on the left side

of face during a transfer operation Immediate blepharospasm (30-min delay in irrigating the exposed areas with water); minor skin burns and irritation to the right eye; severe damage to the left eye (no details provided) Diminished vision in left eye at 3 d; loss of sensation resulting in accidental scratching of the sclera at 3 mo; 1 yr following exposure, the left eye perceived only light and the cornea was vascularized and

opaque Helmers et al., 1971

### C-17 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 20-yr-old male Safety valve release in a

refrigeration unit due to accidental heating of the ammonia line Loss of consciousness, pinkish foaming at the nose and mouth, cyanotic, first- and second-degree burns to the neck, eyes, left arm, glans penis, scrotum, and both lower legs, spastic extremities, both lungs with fine and harsh rales, constant and profuse pulmonary excretions Six-mo followup: normal vision, mild cough White, 1971

20-yr-old female Exposure (approximately

30 min) to anhydrous ammonia following train derailment Chemical burns to the soft palate, oropharynx, and feet, unable to speak, blood-streaked sputum Widespread infiltration consistent with bronchopneumonitis developed shortly after exposure; approximately 1.5–2 yrs following exposure, patient developed a productive cough and was increasingly short-of- breath, bronchiectatic changes involving entire left lung, mild changes in right lung Kass et al., 1972

22-yr-old female Exposure (approximately

90 min) to anhydrous ammonia following train derailment Loss of consciousness, convulsions, chemical burns over the abdomen, left knee and forearm, both arms, soft palate, and oropharynx, damage to both corneas, respiratory distress, multiple areas of alveolar type of infiltrate in both lungs Re-hospitalized 1 yr later with bilateral pneumonia, peripheral edema, and acute right heart failure; marked deterioration of vision in the left eye, bilateral corneal opacities and early cataract changes were present; 2 yrs later, progressive development of infiltrations in basilar section of the lower left lung, generalized varicose bronchiectatic changes in lung segments, hypoxemia, biopsies revealed some mucosal surface erosion, areas of atelectasis, emphysema, alveolar walls thickened with monocellular infiltrates Kass et al., 1972

### C-18 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 24-yr-old male, instrument artificer, smoker Vent stack overflow from

100 feet above work area, resulting in complete drenching Dyspnea, chest pain, blepharospasm, deep cyanosis, burning throat, blisters in the mouth and throat, and congested eyes, sloughs of oral mucosa and exposed skin, conjunctivitis, bronchitis 26 mo following exposure, dyspnea on exertion, excessive collagenization in the bronchial submucosal layer Walton, 1973

39-yr-old male, smoker Vent stack overflow from

100 feet above work area, resulting in complete drenching Dyspnea, chest pain, blepharospasm, burning throat, extensive blistering, tachycardia, moist sounds from both lung bases, corneal burns, aphonia (lasting about 10 d) Continued to smoke at follow up; lung function tests (FVC and expiratory volume in 1 sec) well below normal after 2 yrs Walton, 1973

42-yr-old male, maintenance fitter, smoker Exposure due to the

inadvertent resumption of ammonia gas in a high pressure line during valve maintenance Burns to the left eye (no further details) Five-yr followup indicated normal lung function Walton, 1973

39-yr-old male, assistant

process foreman Doused with ammonia

liquid from approximately 130 feet above Pink frothy sputum, cyanosis, bronchospasm, severe burns and blistering on the face, mouth and hands Recurring bronchitis, dyspnea on exertion; in lung function tests, FVC reached near normal 1 yr following exposure; FEV 1 test indicated moderate obstructive airways disease and the gas transfer figure remained below normal Walton, 1973

39-yr-old male,

process foreman, smoker Doused with ammonia

liquid from approximately 130 feet above Pain in eyes and throat, blepharospasm, burns to the eyes, mouth, face, and throat Continued to smoke at follow up; lung function tests showed progressive improvement in ventilation but consistent depression of gas transfer factor Walton, 1973

47-yr-old male, process worker In vicinity of a high

pressure compressor pump burst Pain and tightness of the chest, blepharospasm, bloodstained sputum, burns and blistering of the face, mouth, eyes, throat, and left heel Lung function tests indicate gradual improvement in ventilatory ability, but below normal in gas transfer factor Walton, 1973

### C-19 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference Male, process worker (age not given) In vicinity of a high

pressure compressor pump burst Immediate fatality; autopsy results indicate extensive edema and burns on the mouth, face, trunk, arms, and upper back; airway at the larynx almost completely blocked, greatly distended and congested lungs Not applicable Walton, 1973

28-yr-old male Sprayed in the face when

a hose coupling came loose First- and second-degree burns to the face, nose, and oropharynx mucosa inflamed and sloughing, rhonchi throughout both lung fields, lung congestion, and segmental atelectasis Reassessment at 6 mo revealed normal chest x-ray films but abnormal pulmonary arterial perfusion and indication of partial airway obstruction in the left basal segment; within 2 wks, this increased to total obstruction of the airways to the left basal segment Taplin et al., 1976

25-yr-old male Tank explosion Initial effects: mil d bilateral conjunctival edema, diffuse bilateral wheezing, rhonchi, and rales, burns to an estimated 30% of the skin surface area of the extremities, chest, and genitalia, pulmonary edema; shortly after admission, development of severe respiratory distress with production of large amounts of mucopurulent exudate Arterial hypoxemia persisted despite respirator inspired oxygen at 50–60% and positive end expiratory pressure; wheezing and bronchospasm, bilateral infiltrates, fatality occurred at 60 d following exposure with evidence of purulent cavitary pneumonia Sobonya, 1977

6-mo-old male; two 12-yr-oldmales; 17-yrold female Road accident involving

a tank truck rolling from an overpass to the road below, resulting in a thick cloud of ammonia

vapor All four victims suffered varying degrees of burns to the face and body,

eyes, mouth, oral mucosa, erythema, and edema of the epiglottis and larynx; two victims required intubation for upper airway obstruction Measurement of several urinary metabolites of hydroxylysine indicated that considerable collagen degradation occurred after inhaling concentrated ammonia vapors Hatton et al., 1979

### C-20 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 17-yr-old female Direct exposure to high

concentrations resulting from a tanker truck explosion Second-degree burns of the forehead and upper extremities, total epithelial loss of the corneas, second- and third-degree burns of the nasal passages, soft palate, posteriorpharyngeal wall and fullthickness burns of the larynx, diffuse parenchymal densities in both lungs, inspiratory wheezing, rhonchi, and rales were present Pulmonary function studies (1 yr following discharge) indicated moderate obstructive and restrictive abnormalities Close et al., 1980

6-mo-old male Direct exposure to high

concentrations resulting from a tanker truck explosion Second- and third-degree burns on the face, scalp, upper extremities, and buttocks, epithelial defects in both corneas, upper airway obstruction (requiring intubation), diffuse erythema and edema of the lips, soft palate, posterior pharyngeal wall, and epiglottis No remarkable findings in 2-mo followup Close et al., 1980

26-yr-old male Direct exposure

(approximately 20 min) to high concentrations resulting from a tanker truck explosion Upper airway obstruction (requiring mechanical ventilation), extensive second-degree burns of the head, neck, and chest, total loss of corneal epithelium of both eyes, extensive second- and third-degree burns of the oropharynx, hypopharynx, and larynx, diffuse rales, rhonchi, and expiratory wheezing, bilateral patchy pulmonary infiltrates Rapid degeneration and fatality followed a massive hemorrhage around the tracheostomy tube (innominate artery erosion);autopsy revealed fullthickness burns to the entire respiratory tract, acutenecrotizing tracheabronchitis, and bilateral bronchopneumonia Close et al., 1980

### C-21 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 23-yr-old female Within a few hundred

feet of the tanker truck explosion, exposure time >30 min Mild facial erythema and bilateral conjunctival irritation, first- and second-degree burns of the oral cavity, oropharynx, and larynx, bilateral parenchymal infiltrate, mild hypoxemia After a period of stability, progressive bilateral pneumonia with progressive dyspnea, and increased secretions; subsequent followup examinations revealed accentuated interstitial markings, profuse, diffuse combined bronchoalveolar and perfusion abnormalities, development of large pneumatoceles, and bullous changes to the right side of the chest with herniation to the left, facial edema, and anasarca; ventilator assistance through a tracheostomy required Close et al., 1980

30-yr-old female In the vicinity of the

tanker truck explosion, exposure time approximately 30 min Mild facial erythema, conjunctival irritation of the eyes, diffuse bilateral rales and rhonchi, bilateral infiltrates, hypoxemia (requiring mechanical ventilation) After initial improvement, pneumonia developed in the lower left lung, accompanied by severe progressive hoarseness, moderate hypoxemia and development of a combined obstructive and restrictive pulmonary disorder; additional 2-yr followup indicated scattered parenchymal scarring, bleb formation in the left lung base and mild to moderate hypoxemia Close et al., 1980

### C-22 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference

Fourteen male fishermen, 18– 39 yrs old Refrigeration system leak

below decks Inflammation of the pharynx and conjunctiva present in 12–14 victims, with 2 showing corneal burns; tachypnea was present in 10 fisherman; tachycardia was seen in 4 fishermen; 5 victims had unremarkable chest examinations upon admission, while 9 were found to have rales, rhonchi, and wheezing, productive cough; 1 of the 9 moderately affected victims developed laryngeal edema requiring tracheostomy, while another had persistent and progressive airway obstruction for 48 hrs Not applicable Montague and Macneil, 1980

58-yr-old female Leaking pipe near the

offices for a grocery distributor Loss of consciousness, severe eye irritation with bilateral corneal burns, dyspnea, pharyngeal edema, bilateral diffuse rhonchi, and rales Two yrs following exposure, patient developed severe pulmonary insufficiency requiring a 1-yr hospitalization with a permanent tracheostomy and continuous oxygen Stroud, 1981

30-yr-old female Exposure to fumes

released when a tanker truck overturned and exploded Initial intense eye and nasopharyngeal irritation developing to breathlessness and nonproductive cough, initial transient infiltrate of pulmonary edema Following apparent recovery from initial acute effects, development of purulent cough with progressive hypoxemia and hypercapnia requiring tracheotomy and mechanically assisted respiration for the next 3 yrs; development of bilateral, severe, cylindrical, and saccular bronchiectasis with obliterative fibrous adhesions of the rightpleural space, bronchopneumonia; fatality Hoeffler et al., 1982

### C-23 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 50-yr-old male Splashed with liquid

ammonia when a refrigeration coolant tank exploded Burns to the right foot and second-degree burns to the thigh and groin, progressive hypoxemia and inspiratory stridor, hyperemic, edematous pharynx and vocal chords, with the posterior pharyngeal wall and the entire tracheal wall below the vocal chords covered with a yellow purulent pseudomembrane Progressively worsening respiratory distress (mechanical ventilation was required for 10 d), extensive pseudomembrane formationthroughout the tracheabronchial tree, gastrointestinal bleeding, persistent tachypnea and expiratory wheezing (following removal of tracheostomy tube), pneumonitis complications;5-yr followup: asymptomatic except during very strenuous activity Flury et al., 1983

Two male factory workers, smokers, 24 and 40 yrs old Sudden release of

ammonia gas to the factory floor when the pressure valve on a refrigeration unit released Eye and throat irritation, slightly red throat; one patient had burns on the legs and genitals O’Kane, 1983

Three male factory workers, smokers, 20– 30 yrs old, with histories of asthma, smoker’s cough, and chronic bronchitis Sudden release of

ammonia gas to the factory floor when the pressure valve on a refrigeration unit released Moderate dyspnea, labored breathing, sore eyes, moderate to severe throat pain, cough,

nausea O’Kane, 1983

30-yr-old male factory worker, smoker, no history of lung problems Sudden release of

ammonia gas to the factory floor when the pressure valve on a refrigeration unit released; exposure time of 2–3 min with full inhalation Severe cough, massive nasal discharge, very sore throat, constant retching, development of inspiratory and expiratory rhonchi Hoarseness and productive cough persisting for several months O’Kane, 1983

28-yr-old male factory worker, smoker, no history of lung problems Sudden release of

ammonia gas to the factory floor when the pressure valve on a refrigeration unit released; directly in line with escaping gas Severely inflamed eyes, lips, tongue, edematous pharynx and hoarseness, high-pitched inspiratory and expiratory rhonchi Not applicable O’Kane, 1983

### C-24 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 44-yr-old male factory worker, nonsmoker, no history of lung problems Sudden release of

ammonia gas to the factory floor when the pressure valve on a refrigeration unit released; 4–5-min

exposure Cyanosed, respiratory distress, bilateral inspiratory and expiratory rhonchi, inflamed eyes, tongue, pharynx; 2 hrs following admission, developed severe dyspnea, central cyanosis, and an audible gurgle from bronchial secretions Following initial improvement, the patient began deteriorating the 13 th d following exposure, lungs developed multiple cavitating lesions, secondary infection, and finally necrotizing pneumonia; patient survived with chronic infective lung disease O’Kane, 1983

25-yr-old male refrigeration technician Sudden, massive leak

from the refrigeration plant Loss of consciousness, severe burns of the face, eyes, mouth, and throat; development of clinical and radiologic features of pulmonary edema with audible crackles over the lung fields and production of large amounts of mucous Respiratory failure with tachypnea and arterial hypoxemia developed; death from respiratory failure 12 wks following exposure; intense congestion of the mucosal surfaces of the trachea and major bronchi; the cut surface of the left lung was crepitant, and cylindrical bronchiectasis was found in the middle and lower lungs Price, 1983

Two males, 26 and 40 yrs old Pipe valve failure

allowing ammonia gas to fill a factory area Mild pharyngitis andconjunctivitis; the 40-yrold male also had burns to legs and genitals Followup every 6 mo for up to 2 yrs: no additional findings Ward et al., 1983

Three males, 26, 36, and 45 yrs old Pipe valve failure

allowing ammonia gas to fill a factory area Moderate conjunctivitis, moderate to severe pharyngitis, tachycardia, and tachypnea, frothy sputum, moderate dyspnea, nausea Followup every 6 mo for up to 2 yrs: impaired pulmonary function at discharge (all three men); two men showed improvement while the third did not; all three men had preexisting lung disease Ward et al., 1983

30-yr-old male Pipe valve failure

allowing ammonia gas to fill a factory area Severe dyspnea, sore eyes, nausea, generalized rhonchi, leukocytosis, and marked hypoxemia Improvement in lung function tests was seen during followup; however, patient experienced dyspnea upon exertion and chronic productive cough Ward et al., 1983

27-yr-old male Pipe valve failure

allowing ammonia gas to fill a factory area Severe dyspnea, sore eyes, nausea, generalized rhonchi, leukocytosis, and marked hypoxemia A right basal consolidation developed requiring prolonged treatment with oxygen; improvement in lung function was seen after 30 d; however, patient experienced dyspnea upon exertion and chronic productive cough Ward et al., 1983

### C-25 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 44-yr-old male Pipe valve failure

allowing ammonia gas to fill a factory area Severe dyspnea, sore eyes, nausea, generalized rhonchi, leukocytosis, and marked hypoxemia Following initial improvement from admission, a relapse occurred, coincident with development of pneumonia, requiring artificial respiration and tracheostomy; during 2-yr followup, patient remained severely impaired in pulmonary function Ward et al., 1983

33-yr-old male Explosion while

transferring ammonia from one tank to another Burns over 12% of total body surface area and oropharynx, bilateral corneal clouding, severe respiratory distress Fatality due to cardiac arrest; autopsy findings: tracheobronchial ulcerations, denuded epithelium, congested lungs, and necrotizing bronchitis with ulceration and membrane formation, micro abscesses of the kidneys Arwood et al., 1985

51-yr-old male smoker Explosion while

transferring ammonia from one tank to another Burns over 50% total body surface area (eyes, back, legs, and oropharynx), severe respiratory distress (bilateral wheezing and bilateral lower lobe infiltrates) Progressive decline/respiratory distress following initial improvement, fatality due to cardiac arrest; autopsy findings: tracheobronchial ulcerations, denuded epithelium, congested lungs, and necrotizing bronchitis with ulceration and membrane formation, medullary fibrosis and tubal necrosis of the kidneys Arwood et al., 1985

Five males, 26– 48 yrs old An overturned tanker

truck released a dense cloud of ammonia gas Four immediate fatalities; autopsy findings: second-degree burns on exposed skin, hyperemic tracheobronchial mucosal surfaces, edematous and congested parenchyma, sloughing of the tracheobronchial epithelium and extensive edema and congestion of the lung parenchyma One delayed fatality (2.5 wks after exposure) due to bronchopneumonia and necrotizing tracheobronchitis Burns et al., 1985

### C-26 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference Twenty two seafood processing plant workers Liquefied ammonia tank

explosion within an enclosed area Fourteen cases with irritative symptoms only; six workers exhibited transient bronchospasm and edema of the lips and oral mucosa Five workers exhibited eye pain and photophobia, four of which developed corneal damage; one fatality due to respiratory failure (2 wks following exposure); one worker developed restrictive lung disease and chronic bronchitis (6-mo followup) Yang, 1987

37-yr-old male with myotonic dystrophy Ammonia intoxication

37 yrs after ureterosigmoidostomy Lethargy, restlessness, irritability, and confusion, both lungs were underinflated due to high diaphragm, severemetabolic encephalopathy, right-sided hydronephrosis (left kidney absent), blood ammonia levels were markedly elevated. None Gilbert, 1988

Four males, all smokers (ages not given) Acute exposure (no

additional details provided) Skin blisters, ocular and nasal burning, dyspnea, chest pain, chest tightness, nonproductive cough, and hemoptysis Wheeze, productive cough, dyspnea persisting for 12– 32 mo; pulmonary function tests indicated reversible airway obstruction in one patient with persistent hemoptysis; submucosal inflammation, denuded epithelium, and thickening of the basement membrane Bernstein and Bernstein, 1989

28-yr-old male Equipment failure while

spraying anhydrous ammonia on farmland Skin burns of 12% total body surface area including face, chest, neck, and right upper extremity; periorbital edema with the right eye swollen shut; edema of the uvula, soft palate, tonsilar pillars, and epiglottis with laryngeal burns, thick mucoid rhinorrhea, and bilateral rhonchi Thirteen mo following exposure, patient presented with perihilar adenopathy and a right paratracheal mass, as well as mild obstructive disease; at 4 yrs postexposure, there was no evidence of paratracheal or hilar masses Millea et al., 1989

### C-27 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 17-yr-old male, farm supply store employee A connector valve

opened on a hose carrying ammonia Partial thickness burns on 18% of total body surface area including the face, chest, and lower extremities; full thickness burns on approximately 3% of total body surface area, corneal burns to the right eye, hoarseness, erythema and edema of the oral mucosa and uvula On postexposure d 5, a pneumomediastinum and subcutaneous emphysema were noted, as well as vocal cord and tracheobronchial edema Millea et al., 1989

22-yr-old male farmer Sprayed while applying

anhydrous ammonia to a field Partial thickness burns over 12% total body surface area of the chest, abdomen, and upper extremities; no pulmonary effects Patient was readmitted 6 d following discharge for an infected burn wound on the right arm Millea et al., 1989

21-yr-old male refrigeration plant worker Directly below

refrigeration pipe rupture Cardiorespiratory arrest; third-degree burns to 20% of the body surface, conjunctivitis, burns of the oral mucosa with swelling of the epiglottis, pulmonary edema and bronchial inflammation Fatality 13 d after exposure due to development of treatment-resistant bronchopneumonia Weiser and Mackenroth, 1989

35-yr-old male refrigeration plant worker In vicinity of

refrigeration pipe rupture Second- degree burns over 10% of total body surface area, dyspnea, nausea, severe eye irritation, nose and throat irritation, pulmonary edema Not applicable Weiser and Mackenroth, 1989

Six refrigeration plant workers, male and female, 19– 24 yrs old In vicinity of

refrigeration pipe rupture Erythema, small burn areas, irritation of the eyes, nose, and throat, dyspnea and nausea to varying degrees Not applicable Weiser and Mackenroth, 1989

Workers at a fertilizer plant Leak and explosion of an

ammonia tank at a fertilizer plant Seven fatalities, 57 injuries (no further details provided) Not applicable Andersson, 1991

### C-28 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 28-yr-old male Tank explosion in an

industrial butter plant Bilateral corneal scarring; first- and second-degree burns of the chest and hands, pharyngeal and laryngeal edema with exudative lesions, crackles and wheezes at the lung bases; production of copious bronchial secretions At 1 wk followup: severe tracheobronchial damage with diffuse erythema, inflammation of the airway walls, hemorrhagic areas and abundant purulent secretions, with severe airflow blockage; during 10 yrs of followup, severe fixed airway obstruction remained, with coexisting bronchiectasis Leduc et al., 1992

30-yr-old male, smoker Occupational exposure to

a refrigeration gas leak in adjacent room; approximately 15-min

exposure Immediate burning of the eyes, upper airways, and skin, with cough and pleuritic chest pain, conjunctivitis and rhinopharyngitis Following discharge from hospital, persistent and progressively worse dyspnea, wheezing on exertion, and atypical chest pain; clinical diagnosis of restrictive lung disease with symptoms ongoing at 2 yrs and beyond de la Hoz et al., 1996

27-yr-old male, smoker Occupational exposure to

a refrigeration gas leak in a poorly ventilated room; approximately 1.5–2 min of additional exposure after detecting a strong odor Difficulty breathing; irritation of the eyes, nose, throat, burning sensation on the skin; respiratory failure requiring mechanical ventilation At 26 mo following exposure, victim had persistent productive cough, dyspnea on exertion and expiratory wheezing; pulmonary function testing showed severe obstructive impairment unresponsive to bronchodilation de la Hoz et al., 1996

46-yr-old male Sprayed while unloading

tanks of ammonium hydroxide (29.4% ammonia) Mucocutaneous burns (unspecified), productive cough, dyspnea and wheezing During 2 yrs following initial treatment and discharge, persistent dyspnea at rest and exertion, frequent episodes of dry cough, wheezing, increasing dyspnea and parethesia of the hands and feet; tests indicated a diagnosis of laryngotracheobronchitis and possible pneumonitis, with evidence of nonspecific bronchial hyper-reactivity and small airway disease de la Hoz et al., 1996

### C-29 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 41-yr-old male, fish-processing plant Several hours spent in the

vicinity of an ongoing ammonia leak from a refrigeration unit, without respiratory protection Eye and nasal irritation and mild facial burning Nasal congestion and intermittent epi staxis, which resolved in about 2 wks, hyposmia (decreased sense of smell), which persisted without additional abnormal findings Prudhomme et al., 1998

51-yr-old maintenance

man Pressurized liquid

ammonia explosion during inspection for maintenance and repair on a refrigerator coolant storage system Examination revealed 28% total body surface area burns to the extremities with some torso involvement; there were no facial burns, erythema, mucosal swelling, or signs of respiratory distress None Sotiropoulos et al., 1998

57-yr-old male, supervisor to above Pressurized liquid

ammonia explosion during inspection for maintenance and repair on a refrigerator coolant storage system Pain in the genital area was first noted an hour after explosion, with significant blistering on the scrotum and distal penis Healing without incident or sequelae Sotiropoulos et al., 1998

28-yr-old male Anhydrous ammonia

explosion at a fertilizer factory Second- and third-degree burns (45% total body surface area) to posterior trunk, buttocks, and bilateral lower extremities, as well as inhalation injury (no additional details provided) No effects observed at 18-mo followup Amshel et al., 2000

47-yr-old male Burst pipe in a liquefied

ammonia production plant, with over 45 min of exposure Mixed burns on the face, neck, chest, genitalia, both lower limbs, and full thickness burns of the back (38% total body surface area), frostbite injury to the back, the eyes showed chemosis and abrasions, congestion and corneal edema increasing with subconjunctival hemorrhaging, harsh breathing sounds with crepitations, intermittent bradycardia Eschar necrosis down to the muscles on the back, persistent intermittent bradycardia and low blood pressure, unusual muscle weakness in all four limbs, fatality due to severe hemorrhage from the endotracheal tube and cardiac arrest George et al., 2000

### C-30 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 25-yr-old male Liquid ammonia spill

during transfer from a barge tank to a dockside tank Facial and neck hyperemia, slightly injected sclera, erythematous petechiae on the right ear, edematous and peeling lips, and bilateral corneal abrasions; vision was blurred and had difficulty breathing Not applicable Latenser and Lucktong, 2000

32-yr-old male Liquid ammonia spill

during transfer from a barge tank to a dockside tank Asymptomatic at the scene; within 90 min developed swollen face and lips, edematous pharynx and vocal compromising 50% of the airway, partial thickness burns on the face, neck, anterior chest, left medial upper arm, both hands, and left anterior thigh, 8– 14% total body surface area with full thickness burns on the anterior thigh Not applicable Latenser and Lucktong, 2000

40-yr-old male Exposed to liquid

ammonia while emptying a refrigeration unit Skin burns of 15% total body surface area to the right neck, torso, arm, and axilla, scattered bilateral rhonchi but no wheezing or stridor, minimal edema and erythema of the larynx and proximal trachea Bilateral infrahilar consolidations and apical infiltrates were noted in postburn d 3; there was no long-term followup Kerstein et al., 2001

Two food processing plant workers, ages not specified Discharge of pressurized

ammonia during maintenance One immediate fatality (pulmonary edema, focal lung hemorrhage); one worker permanently blinded (no additional details) Not applicable Morton, 2005

### C-31 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 32-yr-old male Warehouse explosion

resulting in an ammonia tank rupture Skin burn of 2% total body surface area to the face and scrotum; perforated left tympanic membrane and right corneal injury; initial artificial respiration reinstated within 24 hrs for hypoxia and aspiration Airway sloughing with large amounts of debris requiring tracheostomy; after removal of tracheostomy, patient exhibited dysphagia and hoarseness, evidence of supraglottic and aryenoid edema and left true vocal cord paresis; pulmonary function test at 8 mo indicated obstructive pulmonary disease White et al., 2007

22-yr-old male Warehouse explosion

resulting in an ammonia tank rupture 0.5% total body surface area burns to the scrotum, bilateral corneal injury, tracheobronchitis At 4-wk followup, bilateral uveitis with stromal vascularization, haze and scarring, and pigmented keratic precipitates resulting in legal blindness (after unsuccessful attempt at corneal transplants) White et al., 2007

19-yr-old male Warehouse explosion

resulting in an ammonia tank rupture 0.5% total body surface area burns to the face and scrotum; bilateral corneal injury; tracheobronchitis Peribronchiolar edema was noted 5 d after injury, stridor developed and the patient was reintubated and given artificial respiration; pulmonary function testing at 6 mo showed a fixed obstructive pattern White et al., 2007

21-yr-old male Warehouse explosion

resulting in an ammonia tank rupture 1% total body surface area burns to the face, le ft axilla, and scrotum, bilateral corneal injuries, airway sloughing with large amounts of debris required performing a tracheostomy and mechanical ventilation Extensive peribronchiolar edema, necrosis and sloughing were observed down to the tertiary bronchioles; fatality at 100 d postinjury White et al., 2007

33-yr-old male Warehouse explosion

resulting in an ammonia tank rupture Respiratory difficulty, but no burns to the skin and eyes; peribronchiolar edema limited to the right lung Not applicable White et al., 2007

Patients and hospital staff in the critical care unit Ammonia leak from a

refrigerator Respiratory and ocular irritation Not applicable Price and Watts, 2008

### C-32 DRAFT - DO NOT CITE OR QUOTE

Table C-2. Human case studies and reports of human exposure to ammonia

Case(s) Exposure conditions Immediate effects a Delayed effects b Reference 57-yr-old male, nonsmoker At age 25, massive

accidental exposure to anhydrous ammonia while working in a refrigeration unit Loss of consciousness, corneal scarring, and burns to the face and chest Chronic hypoxemic respiratory failure, bilateral inspiratory crackles, and prolonged expiration, central cylindrical, varicose and cystic bronchiectasis with mild interstitial fibrosis Tonelli and Pham, 2009

20-yr-old male Forklift caught an

ammonia pipe causing a sudden burst of a large quantity of ammonia Loss of consciousness, acute respiratory distress syndrome (which required percutaneous tracheotomy), second and third degree burns of his face, anterior neck, left axilla, left arm, parts of anterior and posterior chest wall, genitalia, and both lower limbs covering 50% total body surface area, symblepharon of both

eyes Continued hospital care for respiratory support until transplantation of both lungs performed 6 mo after

exposure Lalic et al., 2009; Pirjavec et al., 2009

53-yr-old male Forklift caught an

ammonia pipe causing a sudden burst of a large quantity of ammonia Loss of consciousness, acute respiratory distress syndrome Developed obliterating bronchiolitis and restrictive and obstructive ventilation disturbances, unable to work, breathless at rest, lung transplant candidate Lalic et al., 2009

Fifteen males, 31.1 ± 8.8 yrs old Resulted from events

during illicit methamphetamine production, 14/15 due to explosions Burns on body, respiratory distress One fatality, ventilator assistance and skin grafting required Bloom et al., 2008

Five males, 49.4 ± 23.0 yrs old Resulted from farming

accidents, 2/5 due to explosions Burns on body, respiratory distress Ventilator assistance and skin grafting required Bloom et al., 2008

a Effects occurring within 48 hrs of exposure.

b Effects occurring longer than 48 hrs after exposure

. 1

2

### C-33 DRAFT - DO NOT CITE OR QUOTE

C.3. CONTROLLED HUMAN EXPOSURE STUDIES OF AMMONIA INHALATION 1 

### Controlled human exposure studies of inhaled ammoni a are summarized in Table C-3. 2

3 

### Table C-3. Controlled human exposure studies of am monia inhalation

Subjects Exposure conditions Results Reference

Seven male volunteers 500 ppm (354 mg/m 3 ) for

30 min from masked breathing apparatus for nose and throat inhalation.

There was no mention of preexposure examinations Hyperventilation (50–250% increase above controls) characterized by increased breathing rate and expiratory minute volume (i.e., volume of air exhaled in 1 min); no coughing was induced, excessive lacrimation occurred in two subjects; two subjects reported nose and throat irritation that lasted 24 hrs after exposure; no changes were reported in nitrogen metabolism or in blood or urine urea, ammonia, or nonprotein nitrogen. Silverman et al., 1949

Seven male volunteers with an average age of 31

yrs 30, 50, and 90 ppm (21, 35, and

64 mg/m 3 ) for 10 min in an inhalation chamber

Physical and neurological examinations were conducted prior to exposure Increased eye erythema at 90 ppm (64 mg/m 3 ) compared to 30 and 50 ppm (21 and 35 mg/m 3 ) exposure; 90 ppm (64 mg/m 3 ) did not produce significant bronchiospasm or severe lacrimation; intensity of odor perception was reported as higher at 30 and 50 ppm (21 and 35 mg/m 3 ) than at 90 ppm (64 mg/m 3 ) MacEwen et al., 1970

18 healthy servicemen volunteers, 18–39 yrs old 50–344 mg/m 3 (70–486 ppm)

for a half-day (session day 2); sessions on days 1 and 3 acted as controls

All volunteers underwent a preliminary examination prior to exposure No effect at concentrations of 71 mg/m 3 (100 ppm); reduced expiratory minute volume at concentrations ranging from 106 to 235 mg/m 3 (150–332 ppm) compared to controls (not dose dependent); exercise tidal volume was increased at 106 mg/m 3 (150 ppm), but reduced at higher concentrations in a dose-dependent manner Cole et al., 1977

Six male and female volunteers, 24–46 yrs old 25, 50, and 100 ppm (18, 35,

and 71 mg/m 3 ) ammonia for 6 hrs/d 1 time/wk over 6 wks; occasional brief exposure to 150–200 ppm (106–141 mg/m 3 )

There was no mention of preexposure examinations Habituation to eye, nose, and throat irritation after 2–3 wks with short- term adaption; there were no significant differences for common biological indicators, physical exams, or in normal job performance when compared to control subjects; continuous exposure to 100 ppm (71 mg/m 3 ) became easily tolerated and had no effect on general health after acclimation occurred; brief exposure to 150– 200 ppm (106–141 mg/m 3 ) produced lacrimation and transient discomfort Ferguson et al., 1977

15 volunteers, 18– 53 yrs old 50, 80, 110, and 140 ppm (35,

57, 78, and 99 mg/m 3 ) for 2 hrs in an exposure chamber

There was no mention of preexposure examinations. No effect on vital capacity, FEV 1 or forced expiratory volume; 140 ppm (99 mg/m 3 ) caused severe irritation and could not be tolerated; reported eye irritation increased with concentration Verberk, 1977

### C-34 DRAFT - DO NOT CITE OR QUOTE

Table C-3. Controlled human exposure studies of am monia inhalation

Subjects Exposure conditions Results Reference
20 male volunteers; groups of four were exposed to ammonia at various concentrations and durations Group 1: exposed to 2 mg/m 3

(3.0 ppm) for 37 d; Group 2: exposed to 5 mg/m 3 (7.2 ppm) for 17 d; Group 3: exposed to 2 mg/m 3(3.0 ppm) for 35 d with shortterm increases to 10 mg/m 3 (14 ppm); Groups 4 and 5: exposed to 2 and 5 mg/m 3 (3.0 and 7.2 ppm), respectively, for 20 d with variations in temperature and humidity; exposure duration each day was not specified ; there was no mention of preexposure examinations Significantly elevated adrenalin levels in urine at 2.1 mg/m 3 (3.0 ppm); dopamine and DOPA levels in urine were not significantly affected at any concentration; significantincrease of adrenalin and 7-oxycorticosteroids in urine, and 11-oxycorticosteroids free fractions in plasma at 5.1 mg/m 3 (7.2 ppm); increased temperature and humidity resulted in increased urine adrenalin, urine 7-oxycorticosteroids and free 11-oxycorticosteroid levels in plasma at 5.1 mg/m 3 (7.2 ppm) Kalandarov et al., 1984

Unspecified number of volunteer subjects Acute exposure up to 15 sec, 1

time/d at unspecified concentrations; also a separate exposure of 10 inhaled breaths via mouthpiece at unspecified concentrations; there was no mention of preexposure examinations The lachrymatory threshold was 55 ppm (39 mg/m 3 ) and bronchoconstriction was seen at 85 ppm (60.1 mg/m 3 ) Douglas and Coe, 1987

Six healthy volunteers (two males and four females, 25–45 yrs old) and eight volunteers with mild asthma (four males and four females, 18–52 yrs old) 16–25 ppm (11–18 mg/m 3 ) for

30-min sessions with 1 wk between sessions

Pulmonary function was measured before and after

exposure No significant changes in pulmonary function in healthy subjects at any concentration; a decrease in FEV 1 and increased bronchial hyperreactivity was reported in asthmatics exposed to dust and ammonia, but not to ammonia alone; exposure to dust alone caused similar effects, suggesting that dust was responsible for the effects. Sigurdarson et al., 2004

12 healthy volunteers (7 females, 5 males) 21–28 yrs old 5 and 25 ppm (4 and 18 mg/m 3 )

for three separate exposures in inhalation chamber for 1.5 hrs resting and 1.5 hrs exercising on a stationary bike; 1–4 volunteers were exposed on each occasion

Lung function and nasal lavage were performed before and after exposure Reported discomfort in eyes, detection of solvent smell, headache, dizziness, and feeling of intoxication were significantly increased at 5 ppm (4 mg/m 3 ); there were no changes in lung function or exhaled nitric oxide levels in exposed individuals; exposure did not result in upper-airway inflammation or bronchial responsiveness Sundblad et al., 2004

### C-35 DRAFT - DO NOT CITE OR QUOTE

Table C-3. Controlled human exposure studies of am monia inhalation

Subjects Exposure conditions Results Reference Healthy male and female volunteers grouped by age, 18– 35 and 45–65 yrs old Repeated 2-sec exposures at

increasing concentrations ranging from 0.9 to 228 ppm (0.6–161 mg/m 3 ) by dynamic olfactometry

There was no mention of preexposure examinations Mean odor detection threshold <20 ppm (14 mg/m 3 ), mean irritation (lateralization) threshold well above 20 ppm (14 mg/m 3 ), dose-response for odor annoyance and irritation; strong olfactory and moderate to strong irritating sensations at >15 ppm (11 mg/m 3 ) Altmann et al., 2006

43 healthy male volunteers age 21– 47 yrs; one group of 30 men not familiar with the smell of ammonia and 10 men exposed to ammonia regularly at the workplace 0, 10, 20, 20 + 2 peak

exposures at 40, and 50 ppm (0, 7, 14, 14 + 2 peak exposures at 28, and 35 mg/m 3 ) on 5 consecutive days for 4 hrs/d in an exposure chamber Subjects familiar to ammonia reported fewer symptoms than naïve subjects; at concentrations ≤ 20 ppm (14 mg/m 3 ), there were no significant differences in symptoms reported between the groups; the perceivedintensity of symptoms was concentrationdependent in both groups Ihrig et al., 2006

25 healthy volunteers (mean age 29.7 yrs), and 15 mild/moderate persistent asthmatic volunteers (mean age 29.1 yrs) 2–500 ppm (1–354 mg/m 3 )

(ocular and nasal exposure) for various durations lasting up to 2.5 hrs

Baseline lung function was recorded prior to exposure Irritation threshold, odor intensity and annoyance were not significantly different between healthy volunteers and asthmatics; nasal irritation threshold = 129 ppm (91 mg/m 3 ); ocular irritation threshold = 175 ppm (124 mg/m 3 ); there were no changes in lung function (FEV 1 ) for subjects in either

group Petrova et al., 2008

24 healthy female volunteers age 18– 45 yrs (mean age 29.9 yrs) 0.03–615.38 ppm (0.02–

435 mg/m 3 ) (nasal exposure) for a maximum of 2 sec

Preexposure measurements included rhinoscopic exam, screening for chemical sensitivities, allergies, respiratory disease, general health, and prior chemical exposure by personal interview Both the static and dynamic methods showed similar averages for detection thresholds for the odor and irritancy of ammonia; mean odor detection threshold of 2.6 ppm (2 mg/m 3 ) (both static and dynamic) and mean irritation thresholds of 31.7 or 60.9 ppm (22 or 43 mg/m 3 ) for static and dynamic methods, respectively Smeets et al., 2007

1

2

### C-36 DRAFT - DO NOT CITE OR QUOTE

C.4. CROSS SECTIONAL STUDIES OF LIVESTOCK FARMERS EXPOSED TO 1



### AMMONIA 2 

### Cross sectional studies of livestock farmers expose d to ammonia are summarized in 3



### Table C-4. 4

5



### Table C-4. Cross sectional studies of livestock fa rmers exposed to ammonia

Subjects Methods Exposure conditions Results Refere nce 27 pig farmers (mean age of 29 yrs) Environmental and

personal exposures were analyzed; lung function was measured on Monday, Tuesday, and Friday Mean exposure to dust =

1.57 mg/m 3 ; endotoxin = 24 ng/m 3 , and ammonia = 5.60 mg/m 3 There was no significant correlation with lung function and exposure to dust or endotoxins; there was a correlation with decreased lung function (5–10%) and exposure to ammonia on the Tuesday testing, but not the Monday or Friday testing; reported respiratory symptoms included cough, phlegm, and wheezing Heederik et al., 1990

29 farm workers; 48 electronic factory workers (controls) 20 pig houses were

monitored for dust and ammonia concentrations; respiratory symptoms were determined by questionnaire; lung function tests were performed; 24 subjects provided blood samples to determine IgE and IgG antibody levels Mean airborne ammonia

concentrations ranged from 1.5 to 13.23 ppm (1–9 mg/m 3 ) and mean dust concentrations ranged from approximately 2 to 21 mg/m 3 Respiratory symptoms included chest tightness, wheeze, nasal and eye irritation (23/29 farm workers); 3/29 farm workers had impaired lung function (decreased FEV 1 and FVC); 3 farmers had IgE antibodies to pig squames or urine; specific IgG antibodies were found in 14 workers to pig squames, and 9 to pig urine, suggesting an allergic

response Crook et al., 1991

### C-37 DRAFT - DO NOT CITE OR QUOTE

Table C-4. Cross sectional studies of livestock fa rmers exposed to ammonia

Subjects Methods Exposure conditions Results Refere nce 102 pig farmers (mean age 39.7 yrs; mean duration of employment of 15.7 yrs) who worked at least half time in a swine confinement building; 51 male dairy farmers (mean age 40.1 yrs; mean duration of employment of 20.3 yrs) and 81 male dairy industry workers (controls; mean age 38.5 yrs; mean duration of employment of 15.7 yrs) Pulmonary function

tests were given to subjects before and after a methacholine challenge; respiratory symptoms were determined by questionnaire Mean total dust level of

2.41 mg/m 3 ; mean airborne ammonia concentration of 8.5 mg/m 3 ; mean personal ammonia exposure of 3.23 mg/m 3 Pig and dairy farmers had higher prevalence of reported cough and morning phlegm; bronchial hyperreactivity to methacholine was higher for pig and dairy farmers compared to controls Choudat et al., 1994

54 male swine producers (mean age = 36.3 yrs; mean duration of employment = 10.7 yrs) Assessment of

respiratory symptoms with questionnaire and lung function

tests Mean contaminant

levels: carbon dioxide = 2,632 ppm (1,861 mg/

m 3 ); ammonia = 11.3 ppm (8 mg/m 3 ); total dust = 2.93 mg/m 3 ; respirable dust = 0.13 mg/m 3 ; endotoxin = 11,332 units/m 3 Exposure to high concentrations of ammonia was associated with chronic cough and bronchitis; incidence of chronic cough was dependent on interaction of ammonia with endotoxin, and respirable dust; ammonia concentrations were not correlated with changes in lung function parameters Zejda et al., 1994

207 males ≥ 18 yrs of age employed at swine farms and spent time in swine confinement buildings (mean years of employment = 9.6); a farm comparison group (nonconfinement production) was included (number not given) Pulmonary function

tests were performed before shift (baseline) and then after a minimum of 2 hrs of exposure; environmental and personal air samples were made for ammonia, carbon dioxide, hydrogen sulfide, carbon monoxide, total and respirable dust Mean personal air

exposure for all subjects: total dust = 4.53 mg/m 3 ; respirable dust = 0.23 mg/m 3 ; total endotoxin = 202.35 EU/m 3 ; respirable endotoxin = 16.59 EU/m 3 ; ammonia = 5.64 ppm (4 mg/m 3 ) Positive correlations were associated with pulmonary function and exposure to total dust, respirable dust, respirable endotoxin, and ammonia; exposure to ammonia concentrations of
≥ 7.5 ppm (5 mg/m 3 ) were predictive of a ≥ 3% decrease in FEV 1 ; the correlation between exposure and decreased pulmonary function was stronger after 6 yrs of exposure Donham et al., 1995

### C-38 DRAFT - DO NOT CITE OR QUOTE

Table C-4. Cross sectional studies of livestock fa rmers exposed to ammonia

Subjects Methods Exposure conditions Results Refere nce 194 Dutch pig farmers (94 with chronic respiratory symptoms, 100 without symptoms) Cross-sectional study

evaluating exposure response relations of exposures to dust, endotoxins, ammonia, and disinfection procedures Estimates of long-term

exposure based on two personal exposure samples (one winter sample, one summer sample); Mean estimated exposure to dust = 2.7 mg/m 3 , endotoxin = 112 ng/m 3 , ammonia = 2 mg/m 3 Chronic respiratory symptoms included cough, phlegm, chest tightness, and wheezing; exposure to dust, endotoxins, and ammonia were not correlated to chronic respiratory symptoms; ammonia exposure and duration of disinfection were correlated with impairment of baseline lung function (decreased FEV 1 , MMEF, and PEF) Preller et al., 1995

151 males ≥ 18 yrs of age employed at swine farms and spent time in swine confinement buildings (mean years of employment = 12.4); a farm comparison group (nonconfinement production) was included (number not given) Followup study from

Donham et al. (1995) previously described; followup measurements taken 48 mo from the initial measurements Mean personal air

exposure for all subjects: total dust = 3.45 mg/m 3 ; respirable dust = 0.26 mg/m 3 ; total endotoxin = 176.12 EU/m 3 ; respirable endotoxin = 11.86 EU/m 3 ; ammonia = 5.15 ppm (4 mg/m 3 ) Swine workers had a mean cross-shift 2% decrease in FEV 1 that was correlated with personal exposure to total dust, total endotoxin, respirable endotoxin, and ammonia Reynolds et al., 1996

196 pig farmers (96 with chronic respiratory symptoms, 100 without symptoms) Pig farmers tested for

lung function and bronchial responsiveness to histamine challenge Estimates of long-term

exposure based on two personal exposure samples (one winter sample, one summer sample); Mean estimated exposure to respirable dust = 2.7 mg/m 3

, endotoxin = 111 ng/m 3

, ammonia = 2 mg/m 3 No association between bronchial responsiveness and exposure to respirable dust, endotoxins, or ammonia; mild bronchial responsiveness was associated with the disinfectant use of quaternary ammonia Vogelzang et al., 1997

171 pig farmers (82 with chronic respiratory symptoms, 89 without) Longitudinal study

for cohort of pig farmers observed over 3 yrs; subjects examined for lung function and tested for bronchial responsiveness to histamine challenge Estimates of long-term

exposure based on two personal exposure samples (one winter sample, one summer sample); Mean estimated exposure to respirable dust = 2.63 mg/m 3

, endotoxin = 105 ng/m 3

, ammonia = 2 mg/m 3 Decreased lung function (FEV 1 and FVC) wasobserved over time; longterm exposure to ammonia was associated with increased bronchial responsiveness to histamine; exposure to respirable dust also caused increased bronchial responsiveness to histamine Vogelzang et al., 2000, 1998

### C-39 DRAFT - DO NOT CITE OR QUOTE

Table C-4. Cross sectional studies of livestock fa rmers exposed to ammonia

Subjects Methods Exposure conditions Results Refere nce Eight healthy male volunteers (23– 28 yrs old) Exposed for 4 hrs at

1-wk intervals to swine confinement buildings Mean airborne ammonia

concentration of 20.7 ppm (15 mg/m 3 ); also exposed to airborne dust, bacteria, endotoxin, and molds Decreased expiratory flows (FEV 1 ), increased neutrophils in the nasal wash and increased white blood cell count Cormier et al., 2000

257 poultry workers (30% women, 70% men); 63 women and 87 men nonexposed blue-collar workers served as control subjects Personal sampling

conducted for total and respirable dust, total and respirable endotoxin, and ammonia; medical evaluations included pulmonary function tests given before and after a work period Mean exposure levels of

poultry workers: ammonia = 18.4 ppm (13 mg/m 3 ); total dust = 6.5 mg/m 3 ; respirable dust = 0.63 mg/m 3 ; total endotoxin 1,589 EU/m 3 (0.16 μ g/m 3 ); respirable endotoxin = 58.9 EU/m 3 (0.006 μ g/m 3 ) Significant cross-shift declines in pulmonary function were reported for poultry workers; concentrations associated with significant pulmonary function deficits were 12 ppm ammonia (8 mg/m 3 ), 2 .4 mg/m 3 total dust, 0.16 mg/m 3 respirable dust, and 614 EU/m 3 endotoxin (0.614

μ g/m 3 ) Donham et al., 2000

Survey of 8,482 farmers and

spouses; exposure study conducted in 102 farmers Exposure study with

survey of respiratory symptoms; personal exposure to total dust, fungal spores, bacteria, endotoxin, and ammonia in 12 tasks were measured in 102 farmers Ammonia

concentrations ranged from 0 to 8.2 ppm (0–6 mg/m 3 ) over the 12 tasks; total dust (0.4– 5.1 mg/m 3 ), fungal spores (0.02– 2.0 10 6 /m 3 ), bacteria (0.2–48 10 6 /m 3 ), endotoxin (0.5– 28/10 3 EU/m 3 [0.05– 2.8 μ g/m 3 ) There was a significant positive correlation between task mean exposures to total dust, fungal spores, and endotoxins and task-specific symptoms; there was no association between exposures to bacteria and ammonia and task specific symptoms; symptoms included eye, nose, and throat irritation, cough, chest tightness, and wheezing Melbostad and Eduard, 2001

13 stable workers (6 males, 7 females) Stable workers were

tested for lung function and nasal lavage was performed to analyze for inflammation markers; tests were performed during two consecutive winters and the interjacent summer Ammonia concentration

was 20–27 ppm (14– 19 mg/m 3 ) in late summer, but was not detected in winter; levels of endotoxin were highest during late summer (15 ng/m 3 ) while levels of 1,3- β -glucan (85 ng/m 3 ) and horse allergen (18,300 U/m 3 ) were highest during the winter Increased PEF-variability in 2/13 workers; eosinophil cationic protein in 3/13 (indicative of bronchial obstruction and allergic inflammation equivalent to allergic asthma); increased myeloperoxidase and lysozyme levels in 9/13 (indicating enhanced activity of neutrophil granulocytes in the airways and enhanced mucosal secretion) Elfman et al., 2009

EU = endotoxin unit (10 EU/ng); MMEF = mean midexpi ratory flow

1

2

### C-40 DRAFT - DO NOT CITE OR QUOTE

C.5. ACUTE AND SHORT-TERM INHALATION TOXICITY STUD IES OF AMMONIA 1



### IN EXPERIMENTAL ANIMALS 2 

### Acute and short-term inhalation studies (exposure d uration of ≤ 30 days) of ammonia in 3 

### experimental animals are summarized in Table C-5. These studies demonstrate that ammonia 4 

### inhalation can produce changes in pulmonary functio n and histopathological changes in the 5 

### respiratory tract. Acute effects may be followed b y chronic respiratory dysfunction 6 

### characterized by secondary bronchitis, bronchioliti s, and bronchopneumonia. In studies of 7



### cardiovascular and/or metabolic effects of acute or short-term ammonia exposure, ammonia 8 

### exposure was associated with bradycardia, arterial pressure variations, and acidosis (as 9 

### evidenced by a decrease in blood pH and an increase in arterial blood pCO 2 

### ). Several studies 10 

### have investigated amino acid levels and neurotransm itter metabolism in the brain of rats and 11 

### mice following acute inhalation exposure to ammonia . It has been suggested that glutamate and 12



### γ -amino butyric acid (GABA) play a role in ammonia-i nduced neurotoxicity. 13

14C-41 DRAFT - DO NOT CITE OR QUOTETable C-5. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceRatsFemale Porton rats(16/group)0 or 141(0 or 200 ppm)Continuousexposure for 4,8, or 12 dHistology of the trachea 4 d: transitional-stratified appearance of theepithelium;8 d: gross change with disappearance of ciliaand stratification on luminal surface;12 d: increased epithelial thicknessGamble and Clough,1976Male OFA rats(27/group)0 or 354(0 or 500 ppm)Continuousexposure for 1–8 wksBody weight, organweights, airway structure,cell population, alveolarmacrophagesNo deaths occurred; decreased foodconsumption and body weight gain; increasedlung and kidney weights; at 3 wks nasalirritation and upper respiratory tractinflammation, but no effect on lower airways;slight decrease in alveolar macrophages; nohistopathological effects seen at 8 wks,suggesting adaptation to exposureRichard et al., 1978aMale and femaleWistar rats(5/sex/group)9,898–37,825(14,000–53,500 ppm)(no mention of controlgroup)10, 20, 40, or60 minClinical signs, pathology,LC50Eye irritation, eye and nasal discharge, dyspnea;hemorrhagic lungs on necropsy;10-min LC50= 28,492 mg/m3(40,300 ppm)20-min LC50= 20,217 mg/m3(28, 595 ppm)40-min LC50= 14,352 mg/m3(20,300 ppm)60-min LC50= 11,736 mg/m3(16,600 ppm)Appelman et al., 1982Male Crl:COBS CD(Sprague-Dawley)rats (8/group)11, 23, 219, and 818(15, 32, 310, and1,157 ppm); arterialblood collected priorto exposure served ascontrol24 hrs Clinical signs, histology,blood pH, blood gasmeasurementNo clinical signs of toxicity, no histologicdifferences in tracheal or lung sections, nochange in blood pH or pCO2, minor changes inpO2Schaerdel et al., 1983Male Crl:COBS CD(Sprague-Dawley)rats (14/group)3, 17, 31, 117, and505 (4, 24, 44, 165,and 714 ppm); arterialblood collected priorto exposure served ascontrol3 and 7 d Hepatic cytochrome P450content andethylmorphine–Ndemethylase activityNo dose-related change in P450 content orenzyme activitySchaerdel et al., 1983C-42 DRAFT - DO NOT CITE OR QUOTETable C-5. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceMale Long Evansrats (4/group)70 and 212(100 and 300 ppm);results were comparedto “control”, but it wasnot clear if the authorswere referring tohistorical orconcurrent controls6 hrs Clinical signs, behavioralobservationDecreased running, decreased activity Tepper et al., 1985Female Wistar rats(5/group)0, 18, or 212(0, 25, or 300 ppm)6 hrs/d for 5, 10,or 15 dBlood ammonia, urea,glutamine, and pH; brainammonia, glutamine;histopathology of lungs,heart, liver, and kidneys(light and electronmicroscopy)Brain and blood glutamine increased; slightacidosis (i.e., decreased blood pH) 212 mg/m3(300 ppm); lung hemorrhage observed in someexposed ratsManninen et al., 1988Female Wistar rats(5/group)0, 18, or 212(0, 25, or 300 ppm)6 hrs/d for 5 d Plasma and brain ammoniaand amino acid analysisIncrease in brain and plasma glutamineconcentrations, increased brain/plasma ratio ofthreonineManninen andSavolainen, 1989Female albino rats(8/group)0, 848–1,068 (1,200–1,510 ppm)3 hrs Mortality, respiratorymovement and O2consumptionNo deaths reported; inhibition of externalrespiration and decreased O2consumptionRejniuk et al., 2007Male SpragueDawley rats(number/group notgiven)Air concentration notgiven; ammonia vaporadded to inspiratoryline of ventilator;controls exposed tosame volume of roomair20 sec Activity of upper thoracicspinal neuronsLower airway irritation, activation of vagalpulmonary afferents and upper thoracic spinalneurons receiving pulmonary sympathetic inputQin et al., 2007a, bC-43 DRAFT - DO NOT CITE OR QUOTETable C-5. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceMale Wistar rats(4/group)0, 92–1,243 (130–1,758 ppm); thepreexposure periodwas used as thecontrol for eachanimal45 min Airway reflexes by thechanges in respiratorypatterns elicited byammonia in either dry,steam-humidified oraqueous aerosol containingatmospheresAmmonia-induced upper respiratory tractsensory irritation is not affected to anyappreciable extent by wet atmospheres (with orwithout aerosol) up to 1,243 mg/m3Li and Pahluhn, 2010Male rats (10/group)0, 848–1,068 (0,1,200–1,510 ppm) atthe beginning and endof the exposureperiod)3 hrs Oxygen consumption Decreased O2consumption Rejniuk et al., 2008MiceMice (20/group,species, sex notspecified)6,080–7,070(8,600–10,000 ppm);no controls10 min LC50LC50= 7,056 mg/m3(9,980 ppm)Silver and McGrath,1948Male Swiss albinomice (4/group)5,050–20,199(7,143–28,571 ppm);no controls30–120 min LC50LC50(30 min) = 15,151 mg/m3(21,430 ppm) Hilado et al., 1977Albino mice (sex notspecified; 6/dose)Air concentration notmeasured; resultswere compared to“control”, but it wasnot clear if the authorswere referring tohistorical orconcurrent controlsContinuously for2 or 5 dRegional brain metabolism(cerebral cortex,cerebellum, brainstem);MAO, enzymes ofglutamate and ã-aminobutyric acid (GABA)metabolism, and (Na+-K+)-ATPase; amino acid levelsin the brainAltered activities of MAO, glutamatedecarboxylase, alanine amino transferase,GABA-transaminase, and (Na+-K+)-ATPase;increased alanine and decreased glutamateSadasivudu et al.,1979; Sadasivudu andMurthy, 1978C-44 DRAFT - DO NOT CITE OR QUOTETable C-5. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceMale Swiss-Webstermice(4/group)Concentrations notgiven; baseline levelsestablished prior toexposure10 min Reflex decrease inrespiratory rate was usedas an index of sensoryirritation; RD50= theconcentration associatedwith a 50% decrease in therespiratory rateRD50= 214 mg/m3(303 ppm)Kane et al., 1979Male albino ICRmice (12/dose)0–3,436(0–4,860 ppm)1 hr (14-d followup)Clinical signs, bodyweight, organ weight,histopathology, LC50Eye and nose irritation, dyspnea, ataxia,seizures, coma, and death; decreased bodyweight and increased liver to body weight ratioin mice surviving to 14 d; effects in the lungincluded focal pneumonitis, atelectasis, andintralveolar hemorrhage; liver effects includedhepatocellular swelling and necrosis, vascularcongestion; LC50= 2,990 mg/m3(4,230 ppm)Kapeghian et al., 1982Male Swiss-Webstermice (16–24/group)0 or 216(0 or 305 ppm)6 hrs/d for 5 d Respiratory tracthistopathologyLesions in the nasal respiratory epithelium(moderate inflammation, minimal necrosis,exfoliation, erosion, or ulceration); no lesions intrachea or lungsBuckley et al., 1984Male albino ICRmice (12/dose)0, 954, 3,097, or 3,323(0, 1,350, 4,380, or4,700 ppm)4 hrs Hexobarbitol sleepingtime, microsomal proteincontent, liver microsomalenzyme activityIncreased hexobarbitol sleeping time(3,097 mg/m3), increased microsomal proteincontent, aminopyrene-N-deethylase and anilinehydroxylase activities (3,323 mg/m3)Kapeghian et al., 1985Male albino ICRmice (12/dose)0, 81, or 233(0, 115, or 330 ppm)4 hrs/d for 4 d Microsomal proteincontent, liver microsomalenzyme activityNo dose-dependent effects on microsomalenzymesKapeghian et al., 1985Male Swiss mice(6/dose)71 and 212(100 and 300 ppm);data collected duringthe 2 d separatingeach ammoniaexposure served as thecontrol baseline6 hrs Clinical signs, behavioralobservationDecreased running, decreased activity Tepper et al., 1985C-45 DRAFT - DO NOT CITE OR QUOTETable C-5. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceMice (4/group) 3, 21, 40, or 78 (4, 30,56, or 110 ppm),lowest measuredconcentration was thenominal control group2 d Responses to atmosphericammonia in anenvironmental preferencechamber with fourchambers of differentconcentrations of ammoniaNo distinguishable preference for, or aversionto, different NH3concentrationsGreen et al., 2008Male OF1 mice(4/group)0, 92–1,243 (130–1,758 ppm); thepreexposure periodwas used as thecontrol for eachanimal45 min Airway reflexes by thechanges in respiratorypatterns elicited byammonia in either dry,steam-humidified, oraqueous aerosol containingatmospheresAmmonia-induced upper respiratory tractsensory irritation is not affected to anyappreciable extent by wet atmospheres (with orwithout aerosol) up to 1,243 mg/m3Li and Pahluhn, 2010RabbitsFemale NewZealand Whiterabbits (7–9/dose)0, 35, or 71(0, 50, or 100 ppm)2.5–3.0 hrsPulmonary functionDecreased respiratory rate at bothconcentrationsMayan and Merilan,1972Rabbits (species,sex, number/dosenot specified)0, 707–14,140(0, 1,000–20,000 ppm)15–180 min Pulmonary function, deathBradycardia at 1,768 mg/m3(2,500 ppm);arterial pressure variations and blood gasmodifications (acidosis indicated by decreasedpH and increased pCO2) at 3,535 mg/m3(5,000 ppm);death occurred at 4,242 mg/m3(6,000 ppm)Richard et al., 1978bNew Zealand Whiterabbits (16 total;8/dose)Peak concentrations:24,745–27,573 mg/m3(35,000–39,000 ppm);concurrent controlstested4 min Pulmonary function, heartrate, blood pressure, bloodgasesLung injury was evident after 2–3 min(decreased pO2,increased airway pressure)Sjöblom et al., 1999C-46 DRAFT - DO NOT CITE OR QUOTETable C-5. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceCatsMixed breed straycats (5/group)0 or 707(0 or 1,000 ppm)10 min Pulmonary function, lunghistopathology on 1, 7, 21,and 35 d postexposurePulmonary function deficits were correlatedwith lung histopathology; acute effects arefollowed by chronic respiratory dysfunction(secondary bronchitis, bronchiolitis, andbronchopneumonia)Dodd and Gross, 1980PigsYoung pigs(2/group)0, 35, 71, or 106 (0,50, 100, or 150 ppm)Continuousexposure for4 wksClinical signs, foodconsumption, body weight,gross necropsy, organweight, histopathologyLethargy and histopathological alterations in thetracheal and nasal epithelium were observed at71 and 106 mg/m3; decreased body weightoccurred at all concentrations (7–19% decreasefrom control)Drummond et al.,1980Male and FemaleBelgian Landracepigs (4/group)0, 18, 35, or 71(0, 25, 50, or100 ppm)6 d Clinical signs, bodyweight, pulmonaryfunctionLethargy and decreased body weight gain (allconcentrations); no effect on pulmonarymicrovascular hemodynamics and permeabilityGustin et al., 1994Belgian Landracepigs (4/group)0, 18, 35, or 71(0, 25, 50, or100 ppm)6 d Clinical signs, bodyweight, neutrophil count,and albumin in nasallavage fluidNasal irritation (increased neutrophils in nasallavage fluid) and decreased body weight gain atall concentrationsUrbain et al., 1994Landrace-Yorkshirepigs (4/group)0 or 42 (0 or 60 ppm) 15 min/d for8 wksThromboxane A2 (TXA2),leukotriene C4 (LTC4),and prostaglandin (PGI2)productionSignificant increases in TXA2 and LTC4, nosignificant effect on PGI2 productionChaung et al., 2008Hybrid gilts (Whitesynthetic Pietrain,white Duroc,Landrace, LargeWhite)(14 pigs/group)<4 (control) or14 (<5 or 20 ppm)15 wks Salivary cortisol, adrenalmorphometry, bodyweight, food conversionefficiency, general healthscores, play behavior;reaction to light and noiseintensity testedconcurrentlyDecreased salivary cortisol, larger adrenalcortices, less play behavior, no measurableimpact on productivity or physiologicalparametersO’Connor et al., 2010C-47 DRAFT - DO NOT CITE OR QUOTETable C-5. Acute and short-term inhalation toxicity studies of ammonia in animalsAnimalConcentration(mg/m3) DurationParameterexamined Results ReferenceCattleMale Holesteincalves(number/group notspecified)0, 35, or 71(0, 50, or 100 ppm)2.5 hrs Respiration rate, clinicalchemistryNo significant effect on respiration, BUN, pH,pO2, or pCO2Mayan and Merilan,1976Brahman/Charolais(group size notreported)<6 (control), 11, 23, or34 (<8 [control], 16,32, or 48 ppm)12 d Behavioral activity, bodyweight, analysis ofbronchioalveolar lavage(BAL) fluid, hematologicalvariables (hemoglobin,mean cell volume, plateletvolume, eosinophils,neutrophils, total whitecellcount, monocytes)Increased lacrimation, nasal secretions,coughing, increased standing (as opposed tolying down), dose related increases inmacrophage activity and neutrophil percentagein BAL fluid indicating pulmonaryinflammation, no effect on hematologicalvariables or body weightPhillips et al., 2010Holstein Friesianand Brown Swiss(10 of each breed)~0, 4, and 15,(0.3 × 10-6, 6, and21 ppm)10 d at eachconcentrationRespiration and pulse rate,blood gas parametersRespiration and pulse rates were higher ininadequately ventilated barns (elevatedammonia and CO2)Sabuncuoglu et al.,20081

### C-48 DRAFT - DO NOT CITE OR QUOTE

C.6. MECHANISTIC STUDIES 1 

### Portions of this appendix were adapted from the Mec hanisms of Action and Genotoxicity 2 

### sections (Sections 3.3 and 3.5) of the Toxicologica l Profile for Ammonia (ATSDR, 2004) under 3 

### a Memorandum of Understanding with ATSDR. 4

5



### C.6.1. Irritation 6 

### As described in Section 4.1, ammonia is an irritant in humans where the primary and 7 

### most immediate effect of ammonia exposure is burns to the skin, eyes, gastrointestinal tract, and 8 

### respiratory tract. Due to its high water solubilit y, ammonia interacts immediately upon contact 9 

### with available moisture in the skin, eyes, oral cav ity, respiratory tract, and mucous membranes to 10 

### form ammonium hydroxide, which is a weak base. Amm onium hydroxide causes the necrosis of 11 

### tissues through disruption of cell membrane lipids (saponification) leading to cellular destruction 12 

### (Jarudi and Golden, 1973). As cell proteins break down, water is extracted, resulting in an 13 

### inflammatory response, which further damages the su rrounding tissues (Amshel et al., 2000). 14 

### The severity of tissue damage is related to the con centration of the hydroxyl ions and the 15 

### duration of exposure (White et al., 2007; Welch, 20 06; Millea et al., 1989). 16

17 

### C.6.2. Gastric Mucosal Damage 18 

### Ammonium ion may also contribute to adverse effects of H. pylori on the stomach. 19 

### H. pylori produces urease, which breaks down urea that is nor mally present in the stomach into 20 

### ammonia (Mégraud et al., 1992; Tsujii et al., 1992a ). An in vitro study that examined the effects 21 

### of ammonia produced by H. pylori on HEp2 cells showed increased cell vacuolation and 22 

### decreased viability of the cells compared to a urea se negative variant of the same cells (Mégraud 23 

### et al., 1992). An in vivo study suggested that amm onia also causes macroscopic gastric lesions 24 

### and increases the release of endothelin-1 and thyro tropin releasing hormone from the gastric 25 

### mucosa, probably via an endothelin-A receptor, whic h exerts ulcerogenic action on the gastric 26 

### mucosa (Mori et al., 1998). Ammonia may also trigg er the release of cysteine proteases in the 27 

### stomach that contribute to the development of gastr ic hemorrhagic mucosal lesions (Nagy et al., 28 

### 1996). Suzuki et al. (2000) reported findings of a n in vitro assay with gastric surface mucous 29 

### cells from mice that demonstrated apoptosis directe d by mitochondrial membrane destruction 30 

### and enhancing activities of caspase-3 and caspase-9 at concentrations of ammonia detected in 31 

### H. pylori infected patients. Neutrophils that migrate to th e gastric mucosa in response to the 32 

### presence of H. pylori may release hypochlorous acid, which can interact w ith ammonium ion to 33 

### produce the powerful cytotoxic oxidizing agent mono chloramine (Murakami et al., 1995). 34 

### Igarashi et al. (2001) suggest that ammonia acceler ates cytokine-induced apoptosis in gastric 35 

### epithelial cells. 36

### C-49 DRAFT - DO NOT CITE OR QUOTE

Tsujii et el. (1992b) studied the mechanism by whic h ammonia damages the gastric 1 

### mucosa by measuring the following: gastric mucosal damage, gastric mucosal hemodynamics, 2 

### the viability and oxygen consumption of cells separ ated from the gastric mucosa, and the effect 3 

### of ammonia on gastric mucosa mitochondrial oxygen c onsumption. For the measurement of 4 

### gastric mucosal damage, ammonia solutions (2 mL; 0, 125, 187.5, and 150 mM) were 5 

### administered to the stomach of male Sprague-Dawley rats (6 rats/group; 220-250 g). Thirty 6 

### minutes later, the ulcer index (defined as the rati o of the ulcerated area to that of the whole 7 

### stomach as measured using an image-analyzer system) was measured. Solutions ≥ 187.5 mM 8 

### ammonia induced significantly larger ulcers and the increase was dose dependent. The effect of 9 

### pH of the initial ammonia solution was measured by repeating the experiment with 250 mM 10 

### glycine-NaOH/250 mM sodium phosphate solution (pH 1 0.3); this solution did not cause any 11 

### significant damage to the mucosa suggesting that th e effect was due to ammonia and not to 12 

### elevated pH. 13 

### The effects of ammonia on gastric mucosal hemodynam ics were measured (5 rats/group) 14 

### after the surgical placement of an optical fiber bu ndle of a reflectance spectrometer in the 15



### forestomach. Rats were administered 2 ml 250 mM am monia/250 mM sodium phosphate 16 

### solution and their hemoglobin concentration and hem oglobin oxygen saturation were measured. 17 

### There was no significant effect of ammonia administ ration on indices of gastric hemoglobin 18 

### concentration or hemoglobin oxygen saturation. 19 

### Cells from the gastric mucosa were separated and is olated. Ammonia (0, 1, 5, or 10 mM 20 

### ammonia in sodium phosphate buffer, pH 7.4) was add ed to the cell cultures and incubated for 2 21 

### hours at 37 oC. Cell viability was measured using trypan blue exclusion. Cell viability and 22 

### oxygen consumption were significantly lower at 5 an d 10 mM ammonia in sodium phosphate 23



### buffer. 24 

### Mitochondria were obtained from the gastric mucosa of five rabbits (2 kg, the strain was 25 

### not specified) after removal of the stomach. Mitoc hondrial suspensions were treated with 0, 26 

### 0.01, 0.1, 1, 5, or 10 mM ammonia/sodium phosphate in Tris buffer. Oxygen consumption was 27



### measured after the addition of α -ketoglutaric acid followed by adenosine diphosphat e. There 28 

### was significant inhibition of oxygen consumption at the lowest ammonia concentration tested 29 

### (0.01%) without the addition of α -ketoglutaric acid or adenosine diphosphate. Oxyge n 30 

### consumption was significantly lower with α -ketoglutaric acid as the substrate at ≥ 1 mM 31 

### ammonia concentrations; with adenosine diphosphate as the substrate, ammonia concentrations 32



### ≥ 0.1 mM were inhibitory. 33 

### In summary, Tsujii et al. (1992b) showed that milli molar concentrations of ammonia 34 

### inhibit cellular respiration and was cytotoxic to g astric mucosal cells. The effects were not due 35

### C-50 DRAFT - DO NOT CITE OR QUOTE

to the elevated pH of the ammonia solutions. The a uthors concluded that ammonia-induced 1 

### gastric mucosal damage may be due to impairment of cellular energy metabolism. 2

3 

### C.6.3. Genotoxicity Studies 4 

### A limited number of genotoxicity studies are availa ble for ammonia vapor; four of the 5 

### available studies were published between 1932 and 1 951. Studies examining in vivo 6 

### genotoxicity are described in Table C-6. Yadav and Kaushik (1997) examined the genotoxic 7 

### effects of ammonia exposure in 22 fertilizer factor y workers exposed to ammonia at ambient 8 

### concentrations of 88.2 μg/m 3 

### ; 42 nonexposed workers served as control subjects. Increased 9 

### frequencies of chromosomal aberrations and sister c hromatid exchanges in lymphocytes were 10 

### observed in exposed workers compared to control sub jects. Frequencies of chromosomal 11 

### aberrations, sister chromatid exchanges, and mitoti c index all increased with increased duration 12 

### of exposure. This study is difficult to interpret because of sma ll samples sizes and confounding 13 

### by smoking and alcohol consumption. In addition, t he levels of ammonia in the plant seemed 14 

### low compared to other fertilizer plant studies (see for example Section 4.1.4; Rahman et al., 15 

### 2007; Ali, 2001; Ballal et al., 1998); the accuracy and reliability of the sampling and 16



### measurement could not be determined. 17

18

### C-51 DRAFT - DO NOT CITE OR QUOTE

Table C-6. In vivo genotoxicity studies of ammonia

Test system Endpoint Test conditions Results a Dose or concentration b Reference

Human lymphocytes Chromosome

aberrations 22 healthy workers

occupationally exposed to ammonia in fertilizer factory (ambient concentration of 88.28 μg/m 3 ); nonexposed factory staff served as control subjects + 88.28 μg/m 3 Yadav and Kaushik, 1997

Human lymphocytes Sister chromatid

exchanges + 88.28 μg/m 3 Yadav and Kaushik, 1997

Swiss albino mice Micronucleus Intraperitoneal injections for

24

− 48 hr expression times + 12.5-50 mg/kg Yadav and Kaushik, 1997 Drosophila melanogaster Dominant lethal

mutations Dominant lethal assay;

inhalation exposure up to 450 ppm (318 mg/m 3 ) ammonia, 6 hrs/d for 5 d + (T) NA Lobasov and Smirnov, 1934

D. melanogaster Sex-linked

recessive lethal mutations Inhalation exposure to ammonia

as vapor at a concentration killing the majority of flies – (T) NA Auerbach and Robson, 1947

D. melanogaster Dominant lethal

mutations Inhalation exposure to ammonia

as vapor at a concentration killing the majority of flies – (T) NA Auerbach and Robson, 1947

a + = positive; − = negative; (T) = toxicity reported ; NA = not available.

b Lowest effective dose for positive results, highest dose tested for negative or equivocal results.

1 

### Positive results were obtained in a single micronuc leus assay with mice (Yadav and 2 

### Kaushik, 1997). One study in Drosophila exposed to ammonia gas was positive for 3 

### mutagenicity; however, survival after exposure was <2% (Lobasov and Smirnov, 1934). Results 4 

### from another study in Drosophila were negative for sex-linked recessive lethal and dominant 5 

### lethal mutagenicity; however, the majority of Droso phila was killed by ammonia treatment 6 

### (Auerbach and Robson, 1947). 7 

### In vitro tests of the genotoxic effects of ammonia have been performed in bacteria and in 8 

### chick fibroblasts (Table C-7). Ammonia (administer ed as vapor) did not induce reverse 9



### mutations in Salmonella typhimurium or E. coli either with or without metabolic activation 10 

### (Shimizu et al., 1985). Demerec et al. (1951) repo rted an increase in reverse mutations in E. coli ; 11 

### however, positive findings were only reported for l evels of ammonia that were toxic, with 98% 12 

### lethality. Chick fibroblasts immersed in buffered ammonia solution were found to have 13 

### increased frequencies of chromosomal aberrations (R osenfeld, 1932). 14

15

### C-52 DRAFT - DO NOT CITE OR QUOTE

Table C-7. In vitro genotoxicity studies of ammoni a

Test system Endpoint Test conditions Results a

Dose c Reference Without activation With

activation b

S. typhimurium (TA98, TA100, TA1535, TA1537, TA1538); E. coli (WP2 uvrA) Reverse

mutation Plate incorporation

assay with ammonia

vapor – – 25,000 ppm (17,675 mg/m 3 ) ammonia vapor Shimizu et al.,

1985

E. coli (B/SD-4 strains) Reverse

mutation, streptomycin resistance Plate incorporation

assay + (T) ND 0.25% ammonia d Demerec et al.,

1951

Chick fibroblasts Chromosomal

aberrations Cultures immersed in

buffered ammonia solution + ND NA Rosenfeld, 1932

a + = positive; − = negative; (T) = toxicity reported ; NA = not available; ND = no data.

b Exogenous metabolic activation used; S9 liver fract ions from male Sprague-Dawley rats pretreated with pentachlorobiphenyl (KC500).

c Lowest effective dose for positive results, highest dose tested for negative or equivocal results.

d Only positive in treatments using toxic levels of N H 3 (98% lethality).

1